CN112424228A

CN112424228A - Novel bispecific agonistic 4-1BB antigen-binding molecules

Info

Publication number: CN112424228A
Application number: CN201980044254.4A
Authority: CN
Inventors: C·克劳斯; C·费拉拉·科勒; C·克雷恩; E·莫斯纳; P·乌马纳
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2018-07-04
Filing date: 2019-07-02
Publication date: 2021-02-26
Also published as: EP3818082A1; WO2020007817A1; JP2021528988A; TW202035447A; US20210253724A1; AR115702A1

Abstract

The present invention provides a novel bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein the Fab fragments (a) and (b) are fused to each other; methods of producing these molecules and methods of using the molecules are also provided.

Description

Novel bispecific agonistic 4-1BB antigen-binding molecules

Technical Field

The present invention relates to a novel bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein the Fab fragments (a) and (b) are fused to each other. The invention further relates to methods of producing these molecules and methods of using them.

Background

4-1BB (CD137) is one of the members of the TNF receptor superfamily, which was originally identified as a molecule that induces expression by T cell activation (Kwon Y.H., and Weissman S.M (1989), Proc. Natl. Acad. Sci. USA 86, 1963-. Subsequent studies demonstrated that 4-1BB in T and B lymphocytes (Snell L.M. et al (2011) Immunol. revision, 244, 197-217; or Zhang X. et al (2010), J.Immunol.184, 787-795), NK cells (Lin W. et al (2008), Blood 112, 699-707), NKT cells (Kim D.H. et al (2008), J.Immunol.180, 2062-2068), monocytes (Kienzle G. and von Kempis J. (2000), int.Immunol.12, 73-82; or Schwarz H. et al (1995), Blood 85, 1043-2001), neutrophils (Heinisch I.V. et al (2000), Eur.J.30, 3441), amoson H. et al (1995), Blood 85, 1043-2001), neutrophils (Heinisch I.V. et al (2000), dendritic cells (Brooks. 4248; endothelial cells from non-hematopoietic sources such as endothelial cells (Snell L. M. J.M. 12, 3446; Shih. 4246; and H. 4248; endothelial cells from endothelial cells such as endothelial cells, H. 42549, H. 549, Bion-4248), circulation 117, 1292-1301). Expression of 4-1BB in different cell types is mostly inducible and driven by various stimulatory signals such as T Cell Receptors (TCR) or B cell receptors and by receptor-induced signaling by co-stimulatory molecules or pro-inflammatory cytokines (Diehl. et al (2002), J. Immunol.168, 3755-containing 3762; von Kempis J. et al (1997), Osteothritis Cartilage 5, 394-containing 406; Zhang X. et al (2010), J. Immunol.184, 787-containing 795).

CD137 signaling is known to stimulate IFN γ secretion and proliferation from NK cells (Buechel C. et al (2012), Eur. J. Immunol.42, 737-. However, CD137 is most characterized as a costimulatory molecule that regulates T cell CD4⁺And CD8⁺TCR-induced activation in the subpopulation. In combination with TCR triggering, agonistic 4-1 BB-specific antibodies enhance T cell proliferation, stimulate lymphokine secretion and reduce the sensitivity of T lymphocytes to activation-induced cell death (Snell l.m. et al (2011) immunol. revision, 244, 197-217). Consistent with these co-stimulatory effects of 4-1BB antibody on T cells in vitro, their administration to tumor-bearing mice resulted in potent anti-tumor effects in a number of experimental tumor models (Melero I. et al (1997), nat. Med.3, 682-. The in vivo exhaustion experiment shows that the CD8 ⁺T cells play a crucial role in the anti-tumor effect of 4-1 BB-specific antibodies. However, other cell types such as DC, NK cells or CD4 have been reported depending on tumor models or combination therapies including anti-4-1 BB⁺T cell contribution (Murillo O. et al (2009), Eur. J. Immunol.39, 2424-.

It appears that the immunomodulatory properties of 4-1BB agonistic antibodies in vivo require the presence of a wild-type Fc portion on the antibody molecule, thereby suggesting that Fc receptor binding is an important event required for the pharmacological activity of such agents, which has been described as an agonistic antibody specific for other apoptosis-inducing or immunomodulatory members of the TNFR superfamily(Li F. and Ravetch J.V. (2011), Science 333, 1030-1034; Teng M.W. et al (2009), J.Immunol.183, 1911-1920). However, systemic administration of 4-1 BB-specific agonistic antibodies with functionally active Fc domains also induced CD8 in the absence of functional Fc receptors in mice⁺T cell expansion associated with reduced or significant improvement in hepatotoxicity (Dubrot j. et al (2010), Cancer immunol. immunother.59, 1223-1233).

Urelumab (BMS-666513, clone 10C7) is an intact human agonistic non-ligand-blocking monoclonal IgG4 antibody that binds to the 4-1BB extracellular domain. It is disclosed as 20H4.9-IgG4 in U.S. Pat. No. 7,288,638. In human clinical trials (clinical trials. gov, NCT00309023 and NCT00612664), Urelumab treatment was administered once every three weeks and for 12 weeks, which induced disease stabilization in patients with melanoma, ovarian cancer, or renal cell carcinoma. However, these experiments were terminated due to two hepatotoxicity-related deaths resulting from the development of antibody-induced hepatitis grade 4 (Simeone e. and Ascierto p.a. (2012), j. immunogenology 9, 241-. Subsequent detailed analysis of clinical safety data indicates that the occurrence of severe transaminase elevation is mainly caused by a given Urelumab dose. Neutropenia, leukopenia and thrombocytopenia of grade 2+ were also observed. Subsequent detailed analysis of clinical safety data indicates that the occurrence of severe transaminase elevation is mainly caused by a given Urelumab dose. In 2012, Urelumab entered the clinical development phase again, but provided that a dose <1mg/kg was taken and administered once every three weeks. The currently recommended dose is 0.1mg/kg administered every three weeks (N.Segal et al (2016), Results From an Integrated Safety Analysis of Urelumab, an Agonist Anti-CD137 Monoclonal Antibody, Clin.cancer Res., published on line at 12/1/2016). In view of dose-limiting toxicity, there is a need for improved antigen binding molecules specific for 4-1BB, which should act only at tumor specific sites to avoid uncontrollable side effects. The bispecific antigen binding molecules of the present invention bind a Fab fragment capable of preferentially binding to a tumor-specific or tumor-associated antigen (target cell antigen) to two Fab fragments capable of binding to 4-1BB agonism. In its particular form, the bispecific antigen binding molecules of the invention may be capable of triggering not only 4-1BB effectively, but also 4-1BB at the desired site with very high selectivity, thereby reducing the adverse side effects observed in conventional monospecific antibodies such as Urelumab.

Disclosure of Invention

The present invention relates to a novel bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable association, wherein the Fab fragments (a) and (b) are fused to each other; methods of producing these molecules and methods of using them are also provided. These bispecific antigen binding molecules are advantageous because they preferentially activate 4-1BB at the site where the target cell antigen is expressed, by virtue of their binding ability to the target cell antigen, and reduce activation at other sites in the body, thereby avoiding the side effects of antibodies specific for only 4-1 BB. They are also characterized by their specific structural features, such as the proximity of two Fab fragments capable of specifically binding to 4-1BB and the target cell antigen, bivalent binding to 4-1BB and monovalent binding to the target cell antigen, respectively, making these bispecific antigen binding molecules very effective.

In one aspect, the invention provides a bispecific antigen binding molecule comprising:

(a) A first Fab fragment capable of specifically binding to 4-1 BB;

(b) a second Fab fragment capable of specifically binding to a target cell antigen;

(c) a third Fab fragment capable of specifically binding to 4-1 BB; and

(d) an Fc domain consisting of a first subunit and a second subunit capable of stable association; wherein the second Fab fragment (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab fragment (a), which in turn is fused at its C-terminus to the N-terminus of the first Fc domain subunit, and the third Fab fragment (C) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit, and wherein in the second Fab fragment capable of specific binding to a target cell antigen, (i) the variable regions VL and VH of the Fab light chain and Fab heavy chain are replaced with each other, or (ii) the constant regions CL and CH1 of the Fab light chain and Fab heavy chain are replaced with each other.

In a particular aspect, there is provided a bispecific antigen binding molecule as described herein, wherein the bispecific antigen binding molecule provides bivalent binding to 4-1BB and monovalent binding to a target cell antigen.

In a further aspect, there is provided a bispecific antigen binding molecule as described above, wherein the Fc domain consisting of the first and second subunits capable of stable binding is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain. In one aspect, in the CH3 domain of the first subunit of the Fc domain, the amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby creating a bulge within the CH3 domain of the first subunit that can be positioned in a cavity within the CH3 domain of the second subunit; and in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is substituted with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable. Accordingly, a bispecific antigen binding molecule is provided wherein a first subunit of the Fc domain comprises a protuberance and a second subunit of the Fc domain comprises a pore according to the protuberance-into-pore method. In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

In another aspect, bispecific antigen binding molecules as described above are provided, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function. In one aspect, the one or more amino acid substitutions are at one or more positions selected from the group consisting of L234, L235, and P329(Kabat EU index numbering). In one aspect, the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, in particular the amino acid mutations L234A, L235A and P329G (numbered according to the Kabat EU index). Specifically, the Fc receptor is an fey receptor and/or the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC).

In one aspect, there is provided a bispecific antigen binding molecule as described above, wherein the first and third Fab fragments capable of specific binding to 4-1bb, (a) and (c) are the same. In a particular aspect, the invention provides a bispecific antigen binding molecule as described above, wherein the first and third Fab fragments capable of specific binding to 4-1BB each comprise: heavy chain variable region (V) _H4-1BB) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and light chain variable region (V)_L4-1BB) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

In a particular aspect, a bispecific antigen binding molecule comprises a first Fab fragment and a third Fab fragment capable of specifically binding to 4-1BB, each comprising: heavy chain variable region (V)_H4-1BB) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 7; and light chain variable region (V)_L4-1BB) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 8. More specifically, the first and third Fab fragments capable of specifically binding to 4-1BB each comprise: heavy chain variable region (V)_H4-1BB) comprising the amino acid sequence of SEQ ID NO 7; and light chain variable region (V) _L4-1BB) comprising the amino acid sequence of SEQ ID NO. 8.

In one aspect, there is provided a bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein the second Fab fragment (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab fragment (a), which in turn is fused at its C-terminus to the N-terminus of the first Fc domain subunit, and the third Fab fragment (C) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit, wherein in the third Fab fragment capable of specific binding to a target cell antigen, (i) the variable domains VL and VH are replaced by each other, or (ii) the constant domains CL and CH1 are replaced by each other, and wherein in the constant domains CL of the first Fab fragment and the third Fab fragment capable of specific binding to 4-1BB, the amino acid at position 124 is replaced by a lysine (K) (numbered according to the KaEU index) and the amino acid at position 123 is replaced by an arginine (R) or a lysine (K) (numbered according to the KaEU index), and wherein in constant domain CH1 of the first and third Fab fragments capable of specifically binding to 4-1BB, the amino acid at position 147 is substituted with glutamic acid (E) (numbered according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbered according to the Kabat EU index).

In a particular aspect, there is provided a bispecific antigen binding molecule as described above, wherein in the second Fab fragment capable of specific binding to a target cell antigen the variable regions VL and VH of the Fab light chain and Fab heavy chain are replaced with each other.

In a further aspect, there is provided a bispecific antigen binding molecule as described above, wherein the second Fab fragment is capable of specifically binding to a target cell antigen selected from the group consisting of: fibroblast Activation Protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, CD33, and PD-L1. In one aspect, the target cell antigen is selected from the group consisting of: fibroblast Activation Protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, and CD 33. In a particular aspect, the target cell antigen is selected from Fibroblast Activation Protein (FAP), carcinoembryonic antigen (CEA), and CD 19. More specifically, the target cell antigen is selected from FAP and CEA. In another aspect, the target cell antigen is PD-L1.

In one aspect, the second Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to Fibroblast Activation Protein (FAP). Accordingly, there is provided a bispecific antigen binding molecule as described above, wherein the second Fab fragment is capable of specifically binding to FAP and comprises:

(a) Heavy chain variable region (V)_HFAP) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and light chain variable region (V)_LFAP) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:13, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or

(b) Heavy chain variable region (V)_HFAP) comprising: (i) CDR-H1 comprising the tklosterdhee amino acid sequence of SEQ ID NO. 15, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO. 16, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO. 17; and light chain variable region (V)_LFAP) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:18, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:19, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In a further aspect, a Fab fragment capable of specifically binding to FAP comprises:

(a) heavy chain variable region (V)_HFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 21; and light chain variable region (V) _LFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 22; or

(b) Heavy chain variable region (V)_HFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 23; and light chain variable region (V)_LFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 24.

In particular, a Fab fragment capable of specifically binding to FAP comprises: heavy chain variable region (V)_HFAP) comprising the amino acid sequence of SEQ ID NO:21, and a light chain variable region (V)_LFAP) comprising the amino acid sequence of SEQ ID NO 22; or heavy chain variable region (V)_HFAP) comprising the amino acid sequence of SEQ ID NO:23, and a light chain variable region (V)_LFAP) comprising the amino acid sequence of SEQ ID NO 24. More specifically, a Fab fragment capable of specifically binding to FAP comprises: heavy chain variable region (V)_HFAP) comprising the amino acid sequence of SEQ ID NO 21; and light chain variable region (V)_LFAP) comprising the amino acid sequence of SEQ ID NO 22.

Furthermore, there is provided a bispecific antigen binding molecule as described above, wherein

(i) The first and third Fab fragments capable of specifically binding to 4-1BB each comprise: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO 7; and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO 8; and is

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 22; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 24.

In another aspect, the present invention provides a bispecific antigen binding molecule, wherein the second Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA).

Accordingly, there is provided a bispecific antigen binding molecule wherein the second Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises:

(a) heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and light chain variable region (V) _LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or

(b) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:33, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:34, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:36, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:37, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38; or

(c) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:42, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:45, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; or

(d) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:50, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 51; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:52, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:53, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; or

(e) The heavy chain canVariable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:115, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:116, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 117; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:118, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:119, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 120; or

(f) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:123, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:124 or SEQ ID NO:125, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 126; and light chain variable region (V) _LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:127 or SEQ ID NO:128, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:129 or SEQ ID NO:130 or SEQ ID NO:131, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 132.

In one aspect, there is provided a bispecific antigen binding molecule as described above, wherein the second Fab fragment is capable of specifically binding to CEA and comprises:

(a) heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or

(b) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:33, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:34, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35; and light chain variable region (V) _LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:36, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:37(vii) an amino acid sequence, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38; or

(d) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:50, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 51; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:52, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:53, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54.

In another aspect, a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA) comprises:

(a) heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 31; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 32; or

(b) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 39; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 40; or

(c) Heavy chain variable region (V)_HCEA) comprising at least about 95%, 96%, 97%, 98% amino acid sequence identical to that of SEQ ID NO. 4799% or 100% identical amino acid sequence; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO 48; or

(d) Heavy chain variable region (V) _HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 56; or

(d) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 56; or

(e) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 121; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 122; or

(f) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 133; and light chain variable region (V) _LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 143; or

(g) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 137; and light chain variable region (V)_LCEA) comprising at least about 95%, 96%, 97%, 98%, 99% or 100% phase with the amino acid sequence of SEQ ID NO. 143The same amino acid sequence.

More specifically, a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA) comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:31, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 32; or heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:39, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 40; or heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:47, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 48; or heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:55, and a light chain variable region (V) _LCEA) comprising the amino acid sequence of SEQ ID NO: 56. Furthermore, there is provided a bispecific antigen binding molecule as described above, wherein the Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO. 121; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 122.

In one aspect, there is provided a bispecific antigen binding molecule as described above, wherein the second Fab fragment is capable of specifically binding to CEA and comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137 or SEQ ID NO 138; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 139, 140, 141, 142, 143 or 144.

Specifically, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 133 and the light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO. 143; or heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:137, and a light chain variable region (V) _LCEA) comprising the amino acid sequence of SEQ ID NO. 143; or the heavy chain mayVariable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:134, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO. 143; or heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:138, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO: 142; or heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:137, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO: 142; or heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:135, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO: 142; or heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 133 and the light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO: 142.

(ii) The second Fab fragment capable of specifically binding to CEA comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO: 31; and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 32; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 40; or a heavy chain variable region VH whose VH comprises the amino acid sequence of SEQ ID NO:47 and a light chain variable region VL whose amino acid sequence comprises SEQ ID NO: 48; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:55 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 56.

Further, there is provided a bispecific antigen binding molecule as described above, wherein (i) the first and third Fab fragments capable of specific binding to 4-1BB each comprise: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO. 8; and (ii) a second Fab fragment capable of specifically binding to CEA comprises: the heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:121 and the light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 122.

In a further aspect, there is provided a bispecific antigen binding molecule as described above, wherein

(ii) The second Fab fragment capable of specifically binding to CEA comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:133 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 143; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:137 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 143; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:134 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 143; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:138 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 142; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:137 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 142; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:135 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 142; or a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:135 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 142.

In a particular aspect, there is provided a bispecific antigen binding molecule, wherein (i) a first Fab fragment and a third Fab fragment capable of specific binding to 4-1BB each comprise: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8; and (ii) a second Fab fragment capable of specifically binding to CEA comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 32. More specifically, the second Fab fragment capable of specifically binding to CEA comprises: the heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:121 and the light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 122.

In another particular aspect, there is provided a bispecific antigen binding molecule, wherein (i) the first and third Fab fragments capable of specific binding to 4-1BB each comprise: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8; and (ii) a Fab fragment capable of specifically binding to CEA comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40. In another particular aspect, there is provided a bispecific antigen binding molecule, wherein (i) the first and third Fab fragments capable of specific binding to 4-1BB each comprise: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO. 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 8; and (ii) a Fab fragment capable of specifically binding to CEA comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:47 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 48.

In another aspect, the present invention provides a bispecific antigen binding molecule, wherein the second Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to CD 19.

In particular, a bispecific antigen binding molecule is provided, wherein a Fab fragment capable of specifically binding to CD19 comprises:

(a) heavy chain variable region (V)_HCD19) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:57, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:58, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 59; and light chain variable region (V)_LCD19) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:60, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:61, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 62.

Specifically, a Fab fragment capable of specifically binding to CD19 comprises: heavy chain variable region (V)_HCD19) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 63; and light chain variable region (V)_LCD19) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 64. More specifically, a Fab fragment capable of specifically binding to CD19 comprises: heavy chain variable region (V) _HCD19) comprising the amino acid sequence of SEQ ID NO:63, and a light chain variable region (V)_LCD19) comprising the amino acid sequence of SEQ ID NO: 64.

In a particular aspect, a bispecific antigen binding molecule is provided, wherein

(i) The first and third Fab fragments capable of specifically binding to 4-1BB each comprise: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 8; and is

(ii) A second Fab fragment capable of specifically binding to CD19 comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO 63 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO 64.

In a further aspect, the present invention provides a bispecific antigen binding molecule wherein the second Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to PD-L1.

In particular, a bispecific antigen binding molecule is provided, wherein a Fab fragment capable of specifically binding to PD-L1 comprises: (a) heavy chain variable region (V)_HPD-L1) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:145, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:146, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 147; and light chain variable region (V) _LPD-L1) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:148, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:149, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 150.

Specifically, a Fab fragment capable of specifically binding to PD-L1 comprises: heavy chain variable region (V)_HPD-L1) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 152; and light chain variable region (V)_LPD-L1) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 153. More specifically, a Fab fragment capable of specifically binding to PD-L1 comprises: heavy chain variable region (V)_HPD-L1) comprising the amino acid sequence of SEQ ID NO:152, and a light chain variable region (V)_LPD-L1) comprising the amino acid sequence of SEQ ID NO: 153.

(ii) A second Fab fragment capable of specifically binding to PD-L1 comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:152 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 153.

According to another aspect of the present invention there is provided an isolated polynucleotide encoding a bispecific antigen binding molecule as described above. The present invention further provides a vector, in particular an expression vector, comprising the isolated polynucleotide of the present invention; and provides a host cell comprising the isolated polynucleotide or vector of the invention. In some aspects, the host cell is a eukaryotic cell, particularly a mammalian cell.

In another aspect, there is provided a method of producing a bispecific antigen binding molecule as described above, comprising the steps of: (i) culturing a host cell of the invention under conditions suitable for expression of the antigen binding molecule, and (ii) recovering the antigen binding molecule. The invention also encompasses bispecific antigen binding molecules produced by the methods of the invention.

The present invention further provides a pharmaceutical composition comprising a bispecific antigen binding molecule as described above and at least one pharmaceutically acceptable excipient. In one aspect, the pharmaceutical composition is for use in treating cancer.

In addition, the present invention encompasses a bispecific antigen binding molecule as described above or a pharmaceutical composition comprising the same for use as a medicament.

In one aspect, there is provided a bispecific antigen binding molecule as described above or a pharmaceutical composition of the invention for use in

(i) The stimulation of the T-cell response is achieved,

(ii) support the survival of the activated T cells,

(iii) the medicine can be used for treating cancer,

(iv) delay cancer progression, or

(v) Prolonging the survival time of cancer patients.

In one aspect, there is provided a bispecific antigen binding molecule as described above or a pharmaceutical composition of the invention for use in the treatment of cancer. In another aspect, the present invention provides a bispecific antigen binding molecule for use in the treatment of cancer as described above, wherein the bispecific antigen binding molecule is administered in combination with a chemotherapeutic agent, radiation therapy and/or other agent for cancer immunotherapy.

In a further aspect, the present invention provides a method of inhibiting the growth of a tumor cell in an individual, comprising administering to the individual an effective amount of a bispecific antigen binding molecule as described above or a pharmaceutical composition of the invention, to inhibit the growth of the tumor cell.

Further, there is provided the use of a bispecific antigen binding molecule as described above for the manufacture of a medicament for the treatment of a disease in a subject in need thereof, in particular for the manufacture of a medicament for the treatment of cancer or an infectious disease, and a method of treating a disease in a subject comprising administering to said subject a therapeutically effective amount of a composition comprising a bispecific antigen binding molecule of the invention, the composition being in a pharmaceutically acceptable form. In a particular aspect, the disease is cancer. In any of the above aspects, the subject is a mammal, particularly a human.

Drawings

Figure 1A shows one example of a bispecific antigen binding molecule of the present invention. The bispecific antigen binding molecule is a huIgG 1P 329GLALA format comprising two anti-4-1 BB Fab fragments (bivalent binding to 4-1BB) and one anti-FAP cross Fab fragment (Fab fragment wherein the VH and VL regions are exchanged) fused at the C-terminus of its heavy chain to the N-terminus of the heavy chain of one of the 4-1BB Fab fragments. This form is referred to herein as the head-to-head (H2H)2+1 form. Large black dots indicate a protuberance into the pore mutation, while small black dots in the CH1/CL domain indicate amino acid mutations that improve the correct pairing of the heavy chain with the anti-4-1 BB light chain.

Figure 1B shows another bispecific antigen binding molecule in the form of huIgG 1P 329GLALA, comprising two anti-4-1 BB Fab fragments (bivalent binding to 4-1BB) and a VH and VL domain capable of specifically binding to FAP fused at the C-terminus of the heavy chain, respectively. This form is referred to herein as the 2+1 VH/VL form and is used herein as a control molecule. Figure 1C shows a bispecific antigen binding molecule in the form of huIgG 1P 329GLALA comprising the VH and VL domains of two anti-4-1 BB Fab fragments (bivalent binding to 4-1BB) and germline control DP47 (non-targeting control). FIG. 1D shows a standard antibody in the form of huIgG 1P 329GLALA, which contains two anti-4-1 BB Fab fragments (bivalent binding to 4-1 BB).

Figure 1E shows another example of a bispecific antigen binding molecule of the present invention. The bispecific antigen binding molecule is a huIgG 1P 329GLALA format comprising two anti-4-1 BB Fab fragments (bivalent binding to 4-1BB) and one anti-human PD-L1 (termed anti-PD-L1) cross Fab fragment (Fab fragment wherein the VH and VL regions are exchanged) fused at the C-terminus of its heavy chain to the N-terminus of the heavy chain of one of the 4-1BB Fab fragments. This form is referred to herein as the head-to-head (H2H)2+1 form. Large black dots indicate a protuberance into the pore mutation, while small black dots in the CH1/CL domain indicate amino acid mutations that improve the correct pairing of the heavy chain with the anti-4-1 BB light chain. FIG. 1F shows a bispecific antigen binding molecule in the form of huIgG 1P 329GLALA, which comprises one anti-4-1 BB Fab fragment (monovalent binding to 4-1BB) and one anti-human PD-L1 (referred to as anti-PD-L1) cross Fab fragment (Fab fragment, in which the VH and VL regions are exchanged). This form is referred to herein as the 1+1 form. Large black dots indicate a protuberance into the pore mutation, while small black dots in the CH1/CL domain indicate amino acid mutations that improve the correct pairing of the heavy chain with the anti-4-1 BB light chain.

FIGS. 2A and 2B relate to the simultaneous binding of bispecific 2+ 1H 2H anti-4-1 BB x anti-FAP huIgG 1P 329GLALA antigen binding molecules to 4-1BB and FAP. Fig. 2A is a pictogram of a measurement setup. Figure 2B shows the simultaneous binding of bispecific H2H anti-4-1 BB x anti-FAP antigen binding molecule (analyte 1) with immobilized human 4-1BB and human FAP (analyte 2).

Figure 3 shows a FRET-based competition assay for assessing bivalent binding of a 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) antigen binding molecule. The interaction between Tb-labeled hu4-1BB-SNAP expressed on transfected Hek cells and d 2-labeled 4-1BB (clone 20H4.9) IgG was competed by the addition of either the unlabeled 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) huIgG1 PGLALA construct (one of the Fab against 4-1BB has no free N-terminus, filled squares) or the unlabeled 2+1 VH/VL (C-terminus) 4-1BB (20H4.9) x FAP (4B9) bispecific construct (two "free Fab" against 4-1BB, open squares). Competition results in a decrease in the TR-FRET signal.

FIG. 4 shows binding to NIH/3T3-huFAP clone 19 cells expressing human FAP. The concentration of 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) antigen binding molecule (black filled circles and lines) or its control molecule was plotted against the geometric mean of the fluorescence intensity (gMFI) of PE conjugated secondary detection antibody. Baseline correction was performed for all values by subtracting baseline values for blank controls (e.g., no primary detection antibody, only secondary detection antibody included). Constructs containing only FAP antigen binding domain (e.g. anti-4-1 BB (20H4.9) x anti-FAP (4B9)2+ 1H 2H huIgG 1P 329glal (black filled circles and lines) and anti-4-1 BB (20H4.9) x anti-FAP (4B9) )2+ 1VH/VL huIgG 1P 329GLAL (grey filled squares and lines)) binds efficiently to FAP expressing cells. It can be seen that the N-terminally fused anti-FAP cross Fab fragment (black filled circles and lines) of anti-4-1 BB (20H4.9) x anti-FAP (4B9)2+ 1H 2H huIgG 1P 329GLAL exhibits higher gMFI and lower EC than the C-terminally fused VH/VL anti-FAP antigen-binding domain (gray filled squares and lines) of anti-4-1 BB (20H4.9) x anti-FAP (4B9) VH/VL 2+1huIgG 1P 329GLAL₅₀。

FIG. 5 shows binding to the reporter cell line Jurkat-hu4-1BB-NFkB-luc2 expressing human 4-1BB (CD 137). The concentration of 2+1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) antigen binding molecule (black filled circles and lines) or control thereof was plotted against the geometric mean of the fluorescence intensity (gMFI) of PE conjugated secondary detection antibody. Baseline correction was performed for all values by subtracting baseline values for blank controls (e.g., no primary detection antibody, only secondary detection antibody included). Anti-4-1 BB (20H4.9) x anti-FAP (4B9)2+ 1H 2H huIgG 1P 329GLAL (black filled circles and lines) bound to 4-1BB similarly to its control anti-4-1 BB (20H4.9) huIgG 1P 329GLAL (grey stars and lines).

FIGS. 6A to 6C show NF-. kappa.B-mediated luciferase expression activity in 4-1 BB-expressing reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2. To test the functionality of the 2+1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) antigen binding molecule (black filled circles and lines) compared to the 2+1VH/VL anti-4-1 BB (20H4.9) x anti-FAP (4B9) antigen binding molecule (gray filled squares and lines) and controls, the molecules were incubated for 5H with the reporter cell line Jurkat-hu4-1BB-NFkB-luc2 in the absence or presence of a cell line expressing human FAP at a ratio of 1: 5. The concentration of 2+1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) antigen binding molecule or control thereof was plotted against the units of emitted light (RLU) measured 5H after incubation and addition of luciferase assay solution. Baseline correction was performed for all values by subtracting the baseline values of the blank control (e.g., no antibody added). FIG. 6A shows 4-1BB activation independent of FAP target, whereby 4-1BB binding induces expression of NF-. kappa.B-controlled luciferase in a reporter cell line in the absence of any FAP-mediated cross-linking. In FIG. 6B, FAP-expressing human melanoma cell line WM-266-4 (intermediate FAP surface expression) was added. WM-266-4 cells expressing FAP resulted in cross-linking of bispecific 4-1BB (20H4.9) xFAP (4B9) antigen binding molecules. Bispecific FAP targeting 2+1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) antigen binding molecules (black filled circles and lines) showed more excellent activation (lower EC50 values), which likely reflects higher affinity for FAP. This result is better seen in fig. 6C, which shows NF κ B-induced luciferase activation in the presence of the high FAP expressing cell line NIH/3T3-huFAP clone 19 (human FAP transgenic mouse fibroblast cell line).

Figure 7 shows binding of the humanized A5B7 huIgG1P329G LALA variant to MKN-45 compared to the binding of the parent murine A5B7 antibody. The antibody was detected with a fluorescently labeled secondary antibody and fluorescence was measured by flow cytometry.

Fig. 8A and 8B show alignment plots of VH amino acid sequence (fig. 8A) and VL amino acid sequence (fig. 8B) of humanized MFE23 antibody variants.

Figures 9A, 9B and 9C show binding of the humanized MFE23 huIgG1P329G LALA variant to MKN-45 compared to the binding of the parental murine MFE23 antibody. The antibody was detected with a fluorescently labeled secondary antibody and fluorescence was measured by flow cytometry. The three panels show low binding, moderate binding and similar binding to the parent MFE23 clone, respectively.

FIGS. 10A to 10D relate to the simultaneous binding of CEA-targeted trimer cleaving 4-1BBL molecules to hu4-1BB and hu (A2-B2) A or hu (NA1) BA. Fig. 10A shows the assay setup. FIG. 10B shows simultaneous binding of 2+ 1H 2H anti-human 4-1BB (20H4.9) x CEA (A5B7) huIgG1P329GLAL A (analyte 1) to immobilized human N (A2-B2) A and human 4-1BB (analyte 2). FIG. 10C shows simultaneous binding of 2+ 1H 2H 4-1BB (20H4.9) x CEA (A5H1EL1D) huIgG1P329GLAL A (analyte 1) to immobilized human N (A2-B2) A and human 4-1BB (analyte 2). FIG. 10D shows simultaneous binding of 2+ 1H 2H 4-1BB (20H4.9) x CEA (MFE23) huIgG1P329GLAL (analyte 1) to immobilized human (NA1) BA and human 4-1BB (analyte 2). The results of duplicate determinations are shown.

The cell surface CEACAM5 expression levels of different clones expressing CEACAM5 used in the binding assay are shown in fig. 11. Chinese hamster ovary cell line CHO-k1(ATCC CRL-9618) was transfected with either cynomolgus monkey CEACAM5(CHO-k1-cynoCEACAM5 clone 8) or human CEACAM5(CHO-k1-huCEACAM5 clone 11, clone 12 and clone 13). Expression levels were determined by titration of APC-labeled anti-CD 66 specific detection antibody (clone cd66ab.1.1) using flow cytometry. The median of the fluorescence intensity is shown relative to the concentration of the detection antibody, whereby the median of the fluorescence intensity is positively correlated with the amount of detection antibody bound and therefore with the level of expression of CEACAM5 molecules on the cell surface. CHO-k1-cynoCEACAM5 clone 8 and CHO-k1-huCEACAM5 clone 11 showed similar cell surface CEACAM5 expression, while CHO-k1-

huCEACAM5 clones

12 and 13 showed high cell surface CEACAM5 expression levels.

FIGS. 12A to 12D show binding to CHO-k1 cells expressing either Macacam fascicularis CEACAM5 or human CEACAM 5. The concentration of 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA (4B9) antigen binding molecule or control molecule was plotted against the median fluorescence intensity of the PE-conjugated secondary detection antibody. Baseline correction was performed for all values by subtracting baseline values for blank controls (e.g., no primary detection antibody, only secondary detection antibody included). Constructs containing only CEACAM5 antigen binding domain, such as anti-4-1 BB (20H4.9) x CEA (MFE23) huIgG 1P 329GLALA2+ 1H 2H (black filled circles, dashed line), anti-4-1 BB (20H4.9) x CEA (A5B7) huIgG 1P 329GLALA2+ 1H 2H (black filled diamonds, black bars) and 4-1BB (20H4.9) x (A5H1EL1D) huIgG 1P 329GLALA2+ 1H 2H (grey downward triangles, grey bars), binding efficiently to cells expressing human acacem 5 (fig. 12B, 12C and 12D). In contrast, only binding of 4-1BB (20H4.9) x CEA (A5B7) huIgG 1P 329gla 2+ 1H 2H (black filled diamonds, black bars) to cynomolgus CEACAM5 was detected, 4-1BB (20H4.9) x (A5H1EL1D) huIgG 1P 329gla 2+ 1H 2H (grey downward triangles, grey bars) showed only very weak binding to cynomolgus CEACAM5, and 4-1BB (20H4.9) x CEA (MFE23) huIgG 1P 329gla 2+ 1H 2H (black filled circles, dashed lines) showed no binding because MFE23 had no human/cynomolgus cross-reactivity (fig. 12A).

FIGS. 13A to 13D show NF-. kappa.B-mediated luciferase expression activity in 4-1 BB-expressing reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2. To test the functionality of the 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA antigen-binding molecule compared to a control, the molecule was incubated for 5H with the reporter cell line Jurkat-hu4-1BB-NFkB-luc2 in the absence or presence of the CHO-k1 cell line expressing cynomolgus monkey or human CEACAM5 at a ratio of 1: 5. The concentration of 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA antigen binding molecule or control thereof is plotted against the units of emitted light (RLU) measured 5H after incubation and addition of luciferase assay solution. Baseline correction was performed for all values by subtracting the baseline values of the blank control (e.g., no antibody added). All constructs containing the CEACAM5 antigen binding domain relevant to binding assays of CHO-k1 cells expressing CEACAM5, such as anti-4-1 BB (20H4.9) x CEA (MFE23) huIgG 1P 329GLALA 2+ 1H 2H (black filled circle, dashed line), anti-4-1 BB (20H4.9) x CEA (A5B7) huIgG 1P 329GLALA 2+ 1H 2H (black filled diamonds, black lines) and anti-4-1 BB (20H4.9) x (A5H1EL1D) igg 1P 329GLALA 2+ 1H 2H (grey downward triangles, grey lines), induced activation of Jurkat-hu4-1BB-NF κ B-luc2 cell line in the presence of human CEACAM5 expressing CHO-k1 cell line (enhanced C13 and fig. 13D 13). Whereas in the presence of cynomolgus CEACAM5 only anti-4-1 BB (20H4.9) x CEA (A5B7) huIgG 1P 329GLALA 2+ 1H 2H (black filled diamonds, black bars) and anti-4-1 BB (20H4.9) x (A5H1EL1D) huIgG 1P 329GLALA 2+ 1H 2H (grey downward triangles, grey bars) induced Jurkat-hu4-1BB-NF κ B-luc2 reporter cell line activation, but anti-4-1 BB (20H4.9) x CEA (MFE23) huIgG 1P 329GLALA 2+ 1H 2H (black filled circles, dashed lines) did not have this induction since MFE23 has no human/cynomolgus 23 cross-reactivity (fig. 13B). In the absence of CEACAM5 expressing cells, no cross-linking of 4-1BB (20H4.9) x CEA2+ 1H 2H occurred and the Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 reporter cell line was not activated.

Figure 14A shows a setup for assessing simultaneous binding of a PD-L1 targeted 4-1BB agonist construct to hu4-1BB and huPD-L1-Fc. FIG. 14B shows simultaneous binding of 2+ 1H 2H 4-1BB (20H4.9)/PD-L1 human IgG1 PGLALA with huPD-L1-Fc and hu4-1BB-Fc (kih). FIG. 14C shows simultaneous binding of 1+ 1H 2H 4-1BB (20H4.9)/PD-L1 human IgG1 PGLALA with huPD-L1-Fc and hu4-1BB-Fc (kih). Results of three replicate assays are shown.

FIGS. 15A and 15B show binding to MKN45(MKN45-huPD-L1) cells transfected with MKN45 and PD-L1. Geometric mean values of fluorescence intensity of 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-human PD-L1 antigen-binding molecule, 1+1 anti-4-1 BB (20H4.9) x anti-human PD-L1 antigen-binding molecule, or control molecule relative to PE-conjugated secondary detection antibody were plotted. Baseline correction was performed for all values by subtracting baseline values for blank controls (e.g., no primary detection antibody, only secondary detection antibody included). Constructs containing only the PD-L1 antigen binding domain, such as 4-1BB (20H4.9) x PD-L1 huIgG 1P 329glal 2+ 1H 2H (black triangles and black lines) and anti-4-1 BB (20H4.9) x PD-L1 huIgG 1P 329glal 1+1 (grey downward triangles and lines), bind efficiently to MKN45-huPD-L1 cells expressing human PD-L1 (fig. 15B) and not to the parental cell line MKN45 (fig. 15A).

FIGS. 16A and 16B show NF-. kappa.B-mediated luciferase expression activity in 4-1BB expressing reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2. To test the functionality of the 2+ 1H 2H anti-4-1 BB (20H4.9) x PD-L1 and 1+1 anti-4-1 BB (20H4.9) x PD-L1 antigen binding molecules compared to controls, the molecules were incubated for 5H with the reporter cell line Jurkat-hu4-1BB-NFkB-luc2 in the absence or presence of MKN45 cell line expressing human PD-L1 at a ratio of 1: 5. The concentrations of 2+ 1H 2H anti-4-1 BB (20H4.9) x PD-L1 and 1+1anti4-1BB (20H4.9) x PD-L1 antigen binding molecules or controls thereof were plotted against the units of emitted light (RLU) measured 5H after incubation and addition of luciferase assay solution. Baseline correction was performed for all values by subtracting the baseline values of the blank control (e.g., no antibody added). As shown by 1+1anti4-1BB (20H4.9) x PD-L1 huIgG 1P 329GLAL, monovalent binding to 4-1BB results in EC₅₀The value decreased slightly.

Detailed Description

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purpose of interpreting the specification, the following definitions will apply and, where appropriate, terms used in the singular will also include the plural and vice versa.

As used herein, the term "antigen binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins.

As used herein, the term "antigen binding domain capable of specifically binding to a target cell antigen" or "moiety capable of specifically binding to a target cell antigen" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one aspect, the antigen binding domain is capable of activating signaling through its target cell antigen. In a particular aspect, the antigen binding domain is capable of directing an entity (e.g., a 4-1BB agonist) attached thereto to a target site, e.g., to a particular type of tumor cell or tumor stroma that carries an antigenic determinant. Antigen binding domains capable of specifically binding to a target cell antigen include antibodies and fragments thereof as further defined herein. In addition, antigen binding domains capable of specifically binding to a target cell antigen include scaffold antigen binding proteins as further defined herein, e.g. binding domains based on designed repeat proteins or designed repeat domains (see e.g. WO 2002/020565).

With respect to antibodies or fragments thereof, the term "antigen binding domain capable of specifically binding to a target cell antigen" refers to a portion of a molecule that comprises a region that specifically binds to and is complementary to a portion or all of an antigen. An antigen binding domain capable of specific antigen binding can be provided, for example, by one or more antibody variable domains (also referred to as antibody variable regions). Specifically, antigen binding domains capable of specific antigen binding include antibody light chain variable regions (VL) and antibody heavy chain variable regions (VH).

In a particular aspect, an "antigen binding domain capable of specifically binding to a target cell antigen" is a Fab fragment or a cross Fab fragment.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen binding activity.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of a monoclonal antibody preparation, such variants typically being present in minor amounts). In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen.

As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding site binding to the same epitope of the same antigen. The term "bispecific" means that the antigen binding molecule is capable of specifically binding to at least two distinct antigenic determinants. Typically, bispecific antigen binding molecules comprise two antigen binding sites, each of which is specific for a different antigenic determinant. In certain embodiments, the bispecific antigen binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two distinct cells.

The term "valency" as used herein means that a specific number of binding sites specific for a unique antigenic determinant are present in an antigen binding molecule specific for a unique antigenic determinant. Thus, the terms "divalent," "tetravalent," and "hexavalent" indicate the presence of two binding sites, four binding sites, and six binding sites, respectively, in an antigen binding molecule that are specific for a particular antigenic determinant. In a particular aspect of the invention, the bispecific antigen binding molecules according to the invention may be monovalent for a particular antigenic determinant, meaning that they have only one binding site for said antigenic determinant, or may be bivalent or tetravalent for a particular antigenic determinant, meaning that they have two binding sites or four binding sites for said antigenic determinant, respectively.

The terms "full-length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure that is substantially similar to the structure of a native antibody. "native antibody" refers to a native immunoglobulin molecule having a different structure. For example, a natural IgG class antibody is a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two light and two heavy chains that are disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH) (also known as the variable heavy chain domain or heavy chain variable domain) followed by three constant domains (CH1, CH2, and CH3) (also known as heavy chain constant regions). Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL) (also known as a variable light chain domain or light chain variable domain) followed by a light chain constant domain (CL) (also known as a light chain constant region). The heavy chain of an antibody may be assigned to one of five types, referred to as α (IgA), δ (IgD), epsilon (IgE), γ (IgG), or μ (IgM), some of which may be further divided into subtypes such as γ 1(IgG1), γ 2(IgG2), γ 3(IgG3), γ 4(IgG4), α 1(IgA1), and α 2(IgA 2). The light chain of an antibody can be assigned to one of two types, called kappa (. kappa.) and lambda (. lamda.), based on the amino acid sequence of its constant domain.

An "antibody fragment" refers to a molecule other than a whole antibody that comprises a portion of a whole antibody that binds to an antigen to which the whole antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂(ii) a Diabodies, triabodies, tetrabodies, cross-Fab fragments; a linear antibody; single chain antibody molecules (e.g., scFv); and single domain antibodies. For a review of certain antibody fragments, see Hudson et al, Nat Med 9, 129-. For reviews of scFv fragments see, for example, Pl ü ckthun in The pharmacolgy of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab fragments and F (ab')2 fragments that contain salvage receptor binding epitope residues and have extended half-lives in vivo, see U.S. Pat. No.5,869,046. Diabodies are antibody fragments with two antigen binding sites, the diabodiesMay be bivalent or bispecific, see for example EP 404,097; WO 1993/01161; hudson et al, Nat Med 9, 129-; and Hollinger et al, Proc Natl Acad Sci USA 90, 6444-. Trisomal and tetrasomal antibodies are also described in Hudson et al, Nat Med 9,129-134 (2003). A single domain antibody is an antibody fragment comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B 1). Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., e.coli or phage), as described herein.

Papain digestion of whole antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing a heavy and light chain variable domain and a constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Thus, as used herein, the term "Fab fragment" or "Fab molecule" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a light chain constant domain (CL), and a VH domain of a heavy chain and a first constant domain (CH 1). Fab 'fragments differ from Fab fragments in that the Fab' fragment has added to the carboxy terminus of the heavy chain CH1 domain residues that include one or more cysteines from the antibody hinge region. Fab '-SH is a Fab' fragment in which the cysteine residues of the constant domains have a free thiol group. Pepsin treatment to yield F (ab')₂A fragment having two antigen binding sites (two Fab fragments) and a portion of an Fc region. According to the present invention, the term "Fab fragment" also includes "cross-Fab fragments" or "exchanged Fab fragments" as defined below.

The term "crossover Fab fragment" or "crossover Fab molecule" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. Compositions of crossing two different chains of a Fab molecule are possible and are comprised in the bispecific antibody of the invention: in one aspect, the variable regions of the Fab heavy and light chains are exchanged, i.e., the exchanged Fab molecule comprises a peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH1), and a peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL). This exchanged Fab molecule is also known as crossfab (vlvh). On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the exchanged Fab molecule comprises a peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH 1). This exchanged Fab molecule is also known as CrossFab (CLCH 1).

A "single chain Fab fragment" or "scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1(CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, c) VH-CL-linker-VL-CH 1, or d) VL-CH 1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragment is stabilized via the native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single chain Fab molecules may be further stabilized by creating interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

An "exchange-type single chain Fab fragment" or "x-scFab" is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1(CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH 1 and b) VL-CH 1-linker-VH-CL; wherein VH and VL together form an antigen binding site that specifically binds to an antigen, and wherein the linker is a polypeptide of at least 30 amino acids. In addition, these x-scFab molecules can be further stabilized by creating interchain disulfide bonds via insertion of cysteine residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain, according to Kabat numbering).

"Single chain variable fragment (scFv)" is the variable region of the heavy chain (V) of an antibody_H) And light chain variable region (V)_L) The fusion proteins of (a), linked by a short linker peptide of 10 to about 25 amino acids. The linker is generally rich in glycine for flexibility and serine or threonine for solubility, and V may be substituted_HN-terminal of (5) and V_LOr vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original antibody. scFv antibodies are described, for example, in Houston, J.S., Methods in enzymol.203(1991) 46-96). In addition, antibody fragments comprise single chain polypeptides characterized by a VH domain, i.e., capable of assembly with a VL domain to a functional antigen binding site; or a VL domain, i.e. capable of assembling together with a VH domain to a functional antigen binding site, thereby providing the antigen binding properties of a full length antibody.

"scaffold antigen binding proteins" are known in the art, e.g., fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen binding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffold as next-generation antibody therapy. curr Opin Chem Biol 13: 245-. In one aspect of the invention, the scaffold antigen binding protein is selected from the group consisting of: CTLA-4(Evibody), lipocalin (Anticalin), protein a-derived molecules such as the Z-domain of protein a (affibody), a-domain (Avimer/macroantibody), serum transferrin (trans-body); designed ankyrin repeat proteins (darpins), variable domains of antibody light or heavy chains (single domain antibodies, sdabs), variable domains of antibody heavy chains (nanobodies, aVH), V _NARFragments, fibronectin (AdNectin), C-type lectin domains (tetranectin); variable domain (V) of the neoantigen receptor beta-lactamase_NARFragments), human gamma-crystallin or ubiquitin protein (Affilin molecules); human eggKunitz-type domains of protease inhibitors, minibodies (such as proteins from the knottin family), peptide aptamers, and fibronectin (adnectins). CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is predominantly CD4⁺The CD28 family of receptors expressed on T cells. Its extracellular domain has a variable domain-like Ig fold. The loops corresponding to the CDRs of the antibody can be substituted with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as evibods (e.g., US7166697B 1). Evibody is about the same size as the isolated variable region of an antibody (e.g., a domain antibody). For further details, see Journal of Immunological Methods 248(1-2),31-45 (2001). Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules, such as steroids, cholesterol, retinoids, and lipids. They have a rigid β -sheet secondary structure with many loops at the open ends of the cone structure, and can be engineered to bind different target antigens. Anticalin is between 160-180 amino acids in size and is derived from lipocalin. For further details, see Biochim Biophys Acta 1482:337-350(2000), US7250297B1 and US 20070224633. Affibodies are scaffolds of protein a derived from Staphylococcus aureus (Staphylococcus aureus), which can be engineered to bind antigen. This domain consists of a triple helix bundle of about 58 amino acids. Libraries have been formed by randomization of surface residues. For further details, see Protein Eng.Des.Sel.2004,17,455-462 and EP1641818A 1. Avimer is a multidomain protein derived from the a domain scaffold family. The native domain of about 35 amino acids adopts a defined disulfide bonding structure. Diversity is created by natural variation exhibited by the recombinant a domain family. For further details, see Nature Biotechnology 23(12), 1556-. Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered by inserting peptide sequences in permissive surface loops to bind different target antigens. Examples of engineered transferrin scaffolds include the trans body. About For further details, see J.biol.chem 274,24066-24073 (1999). The designed ankyrin repeat protein (DARPin) is derived from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to cell scaffolds. The single ankyrin repeat is a 33 residue motif consisting of two alpha helices and one beta turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and beta-turn in each repeat sequence. Their binding interface can be increased by increasing the number of modules (affinity maturation method). For further details, see J.mol.biol.332,489-503(2003), PNAS 100(4),1700-1705(2003) and J.mol.biol.369,1015-1028(2007) and US20040132028A 1. Single domain antibodies are antibody fragments consisting of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of the heavy chain of an antibody of the camelid family (nanobody or V)_HH fragment). Furthermore, the term single domain antibody comprises an autologous human heavy chain variable domain (aVH) or shark derived V_NARAnd (3) fragment. Fibronectin can be engineered to bind to a scaffold of an antigen. Adnectin consists of a backbone of the native amino acid sequence of domain 10 of the 15 repeat unit of human fibronectin type III (FN 3). The three loops at one end of the β -sandwich can be engineered to enable the Adnectin to specifically recognize a therapeutic target of interest. For further details, see Protein eng.des.sel.18,435-444(2005), US20080139791, WO2005056764, and US6818418B 1. Peptide aptamers are combinatorial recognition molecules consisting of a constant scaffold protein, usually thioredoxin (TrxA), containing a constrained variable peptide loop inserted at the active site. For further details, see Expert opin. biol. ther.5,783-797 (2005). The minibodies are derived from naturally occurring miniproteins of 25-50 amino acids in length containing 3-4 cysteine bridges, examples of which include KalataBI and conotoxins, and knottin. The micro-proteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the micro-protein. For further details on engineered knottin domains, see WO2008098796。

"antigen-binding molecule that binds to the same epitope" as a reference molecule refers to an antigen-binding molecule that blocks binding of the reference molecule to its antigen by 50% or more in a competition assay, and conversely, blocks binding of the antigen-binding molecule to its antigen by 50% or more in a competition assay.

The term "antigen binding domain" or "antigen binding site" refers to a portion of an antigen binding molecule that comprises a region that specifically binds to and is complementary to a portion or all of an antigen. In the case of large antigens, the antigen binding molecule may bind only a specific part of the antigen, which is called an epitope. The antigen binding domain may be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen-binding portion binds, thereby forming an antigen-binding portion-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other diseased cells, on the surface of immune cells, in serum free and/or in extracellular matrix (ECM). Unless otherwise indicated, a protein used herein as an antigen can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human protein. When referring to a particular protein herein, the term encompasses "full-length," unprocessed protein, as well as any form of protein that results from intracellular processing. The term also encompasses naturally occurring protein variants, such as splice variants or allelic variants.

Specific knot"in combination" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding molecule to bind to a particular antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art (e.g., Surface Plasmon Resonance (SPR) techniques (analyzed on a BIAcore instrument) (Liljebllad et al, Glyco J17, 323-329(2000)) as well as conventional binding assays (Heeley, Endocr Res28,217-229 (2002). in one embodiment, the degree of binding of the antigen-binding molecule to an unrelated protein is less than about 10% of the degree of binding of the antigen-binding molecule to the antigen, e.g., as measured by SPR. in some embodiments, the dissociation constant (Kd) of the molecule that binds to the antigen is less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, less than or equal to 0.1nM, less than or equal to 0.01nM, or less than or equal to 0^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M)。

"affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of rate constants remains the same. Affinity can be measured by conventional methods known in the art, including those described herein. A particular method of measuring affinity is Surface Plasmon Resonance (SPR).

An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) which result in an improvement in the affinity of the antibody for an antigen compared to a parent antibody that does not have such alterations.

As used herein, "target cell antigen" refers to an antigenic determinant present on the surface of a target cell, e.g., a cell in a tumor (such as a cell of a cancer cell or tumor stroma). In certain embodiments, the target cell antigen is a tumor specific or Tumor Associated Antigen (TAA). In one embodiment, the TAA is an antigen on the surface of a tumor cell. In one embodiment, the TAA is on a cell of the tumor stroma. In another aspect, the target cell antigen is an antigen on a T cell or a B cell. In one embodiment, the target cell antigen is selected from the group consisting of: fibroblast Activation Protein (FAP), carcinoembryonic antigen (CEA), melanoma-associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), CD19, CD20, CD33, and PD-L1. In a particular aspect, the target cell antigen is Fibroblast Activation Protein (FAP), carcinoembryonic antigen (CEA), or CD 19. More specifically, the target cell antigen is Fibroblast Activation Protein (FAP) or CEA. In another aspect, the target cell antigen is PD-L1.

The term "Fibroblast Activation Protein (FAP)" also referred to as prolyl endopeptidase FAP or Seprase (EC 3.4.21), unless otherwise specified, refers to any native FAP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term includes "full-length" unprocessed FAP, as well as any form of FAP produced by processing in a cell. The term also encompasses naturally occurring variants of FAP, such as splice variants or allelic variants. In one embodiment, the antigen binding molecules of the invention are capable of specifically binding to human, mouse and/or cynomolgus FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884(149 th edition, SEQ ID NO:86) or NCBI (www.ncbi.nlm.nih.gov /) RefSeq NP-004451.2. The extracellular domain (ECD) of human FAP extends from the amino acid at position 26 to the amino acid at position 760. The amino acid sequence of the His-tagged human FAP ECD is shown in SEQ ID NO 87. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321(126 th edition, SEQ ID NO:88) or NCBI RefSeq NP-032012.1. The extracellular domain (ECD) of mouse FAP extends from the amino acid at position 26 to the amino acid at position 761. SEQ ID NO 89 shows the amino acid sequence of the His-tagged mouse FAP ECD. SEQ ID NO:90 shows the amino acid sequence of the His-tagged cynomolgus FAP ECD. Preferably, the anti-FAP binding molecules of the invention bind to the extracellular domain of FAP.

The term "carcinoembryonic antigen (CEA)" also referred to as carcinoembryonic antigen-associated cell adhesion molecule 5(CEACAM5) refers to any native CEA from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise indicated. The amino acid sequence of human CEA is shown in UniProt accession number P06731 (version 151, SEQ ID NO: 91). CEA has long been identified as a tumor associated antigen (Gold and Freedman, J Exp Med., 121: 439-. CEA was originally classified as a protein expressed only in fetal tissues and has now been identified in a variety of normal adult tissues. These tissues are mainly of epithelial origin, including cells of the gastrointestinal, respiratory and genitourinary tracts as well as cells of the colon, cervix, sweat glands and prostate (Nap et al, Tumour biol., 9(2-3):145-53, 1988; Nap et al, Cancer Res., 52(8):2329-23339, 1992). Both epithelial-derived tumors and their metastases comprise CEA as a tumor-associated antigen. The presence of CEA itself does not indicate that it has been transformed into cancer cells, but the distribution of CEA is indicative. In normal tissues, CEA is normally expressed on the apical surface of cells (S) ((R))

S. Semin Cancer biol.9(2):67-81(1999)), rendering it unabsorbable by antibodies in the bloodstream. CEA tends to be expressed on the entire surface of cancer cells compared to normal tissues: (

S., Semin Cancer biol.9(2):67-81 (1999)). This change in expression pattern allows CEA to readily bind to antibodies in cancer cells. Furthermore, expression of CEA in cancer cells is increased. Furthermore, an increase in CEA expression promotes an increase in intercellular adhesion, which may lead to metastasis (Marsha)ll J., Semin Oncol, 30(a suppl.8):30-6,2003). Expression of CEA in various tumor entities is generally very high. Based on published data, the high incidence of CEA has been demonstrated in its own analyses in tissue samples, with an incidence of about 95% in large bowel cancer (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC, co-expressed with HER 3) and 40% in breast cancer; and found to be low in expression levels in small cell lung cancer and glioblastoma.

CEA is readily cleaved from the cell surface and flows from the tumor into the bloodstream, either directly or through lymphatic vessels. Because of this property, serum CEA levels have been used as a clinical index for diagnosing Cancer and screening for recurrence of Cancer, particularly colorectal Cancer (Goldenberg D M., The International Journal of Biological Markers, 7:183-188, 1992; Chau I et al, J Clin Oncol., 22:1420-1429, 2004; Flarni et al, Clin Cancer Res; 12(23): 6985-6986, 2006).

The term "melanoma-associated chondroitin sulfate proteoglycan (MCSP)" is also referred to as chondroitin sulfate proteoglycan 4(CSPG4) and, unless otherwise specified, refers to any native MCSP from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). The amino acid sequence of human MCSP is shown in UniProt accession number Q6UVK1 (version 103, SEQ ID NO: 92). The term "Epidermal Growth Factor Receptor (EGFR)" also known as the protooncogene c-ErbB-1 or the receptor tyrosine protein kinase ErbB-1, unless otherwise specified, refers to any native EGFR from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats). The amino acid sequence of human EGFR is shown in UniProt accession number P00533(211 th edition, SEQ ID NO: 93). The term "CD 19" refers to the B lymphocyte antigen CD19, also known as the B lymphocyte surface antigen B4 or the T cell surface antigen Leu-12, and unless otherwise specified, the term includes any native CD19 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD19 is shown in UniProt accession number P15391 (version 160, SEQ ID NO: 94). "CD 20" refers to the B lymphocyte antigen CD20, also known as transmembrane 4 domain subfamily a member 1(MS4a1), B lymphocyte surface antigen B1, or leukocyte surface antigen Leu-16, and unless otherwise specified, the term includes any native CD20 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD20 is shown in UniProt accession number P11836(149 th edition, SEQ ID NO: 95). "CD 33" refers to the myeloid cell surface antigen CD33, also known as SIGLEC3 or gp67, and unless otherwise specified, the term includes any native CD33 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The amino acid sequence of human CD33 is shown in UniProt accession number P20138(157 th edition, SEQ ID NO: 96).

The term "PD-L1", also known as CD274 or B7-H1, refers to any native PD-L1 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys) and rodents (e.g., mice and rats), particularly "human PD-L1". The amino acid sequence of fully human PD-L1 is shown in UniProt (www.uniprot.org) accession number Q9NZQ7(SEQ ID NO: 106). The term "anti-PD-L1 antibody" or "antibody binding to human PD-L1" or "antibody specifically binding to human PD-L1" or "antagonist anti-PD-L1" refers to an antibody that specifically binds to human PD-L1 antigen with a binding affinity KD value of 1.0X 10^-8mol/L or less, and in one aspect, a KD value of 1.0X 10^-9mol/L or less. Using standard binding assays (such as surface plasmon resonance techniques: (a))

GE-Healthcare Uppsala, sweden)) to determine binding affinity.

The term "variable region" or "variable domain" refers to a domain of an antibody heavy or light chain that is involved in the binding of an antigen binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved Framework Regions (FR) and three hypervariable regions (HVRs). See, e.g., Kindt et al, Kuby Immunology, 6 th edition, w.h.freeman and co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "hypervariable region" or "HVR" as used herein refers to the various regions of an antibody variable domain which are hypervariable in sequence and determine antigen-binding specificity, e.g., "complementarity determining regions" ("CDRs").

Typically, an antibody comprises six CDRs; three in VH (CDR-H1, CDR-H2, CDR-H3) and three in VL (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs herein include:

(a) the hypervariable loops which occur at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2) and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));

(b) CDRs present at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2) and 95-102(H3) (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

(c) antigen contacts present at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2) and 93-101(H3) (MacCallum et al, J.mol.biol.262:732-745 (1996)).

Unless otherwise indicated, the CDRs are determined according to the methods described by Kabat et al (supra). One skilled in the art will appreciate that the CDR names can also be determined according to the methods described by Chothia (supra), McCallum (supra), or any other scientifically accepted nomenclature system. Kabat et al defines a numbering system for the variable region sequences applicable to any antibody. One of ordinary skill in the art can unambiguously assign this "Kabat numbering" system to any variable region sequence, without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" or "Kabat EU index" refers to the numbering system proposed by Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991). Unless otherwise indicated, reference to the numbering of a particular amino acid residue position in the variable region of an antibody is according to the Kabat EU index numbering system.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FRs of a variable domain typically consist of the following four FR domains: FR1, FR2, FR3 and FR 4. Thus, HVR and FR sequences typically occur in the VH (or VL) as follows: FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of antibodies refers to the type of constant domain or constant region that the heavy chain of an antibody has. There are five major classes of antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes may be further divided into subclasses (isotypes), e.g. IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ, and μ, respectively.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. The humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a non-human antibody, in "humanized form" refers to an antibody that has been subjected to humanization. Other forms of "humanized antibodies" encompassed by the present invention are antibodies in which the constant regions have been otherwise modified or altered relative to the original antibody to produce properties according to the present invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.

A "human" antibody is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from a non-human source using a human antibody repertoire or other human antibody coding sequences. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an antibody heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. The IgG Fc region comprises an IgG CH2 domain and an IgG CH3 domain. The "CH 2 domain" of the human IgG Fc region typically extends from amino acid residue at approximately position 231 to amino acid residue at approximately position 340. In one embodiment, the carbohydrate chain is attached to a CH2 domain. The CH2 domain herein may be the native sequence CH2 domain or a variant CH2 domain. The "CH 3 domain" comprises a stretch of residues C-terminal to the CH2 domain in the Fc region (i.e., from amino acid residue at position about 341 to amino acid residue at position about 447 of IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain having an introduced "bulge" ("protuberance") in one chain and a corresponding introduced "cavity" ("pore") in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as described herein. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxy terminus of the heavy chain. However, the antibody produced by the host cell may undergo post-translational cleavage of one or more, in particular one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced by a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may comprise the full-length heavy chain, or the antibody may comprise a cleaved variant of the full-length heavy chain. This may be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, according to the Kabat EU index). Thus, the C-terminal lysine (K447) or C-terminal glycine (G446) and lysine (K447) of the Fc region may or may not be present. The amino acid sequence of the heavy chain comprising the Fc region is represented herein as without the C-terminal glycine-lysine dipeptide if not otherwise indicated. In one aspect, a heavy chain comprising an Fc region as specified herein comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to EU index of Kabat) is comprised in an antibody according to the invention. In one aspect, a heavy chain comprising an Fc region as specified herein is comprised in an antibody according to the invention, the heavy chain comprising an additional C-terminal glycine residue (G446, numbering according to EU index of Kabat). Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"knob-into-hole" techniques are described in, for example, US 5,731,168; US 7,695,936; ridgway et al, Prot Eng 9,617- & 621(1996) and Carter, J Immunol Meth 248,7-15 (2001). In general, the method involves introducing a bulge ("protuberance") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the bulge can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. The bulge is constructed by substituting a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities having the same or similar size as the bulge are created in the interface of the second polypeptide by substituting a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The projections and cavities can be made by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis. In a particular embodiment, the protuberance modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, while the pore modification comprises the amino acid substitutions T366S, L368A and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit comprising a protuberance-modified Fc domain further comprises amino acid substitution S354C, and the subunit comprising a pore-modified Fc domain further comprises amino acid substitution Y349C. The introduction of these two cysteine residues results in the formation of disulfide bridges between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J immunological Methods 248,7-15 (2001)).

"region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin, as well as modified variants having the ability to make substitutions, additions or deletions without substantially reducing the ability of the immunoglobulin to mediate effector functions, such as antibody-dependent cellular cytotoxicity. For example, one or more amino acids may be deleted from the N-terminus or C-terminus of an Fc region of an immunoglobulin without substantial loss of biological function. Such variants may be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J.U. et al, Science 247:1306-10 (1990)).

The term "effector function" refers to those biological activities that can be attributed to the Fc region of an antibody that vary with the isotype of the antibody. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), Antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

Fc receptor binding-dependent effector function can be mediated by the interaction of the Fc region of an antibody with Fc receptors (fcrs), which are specific cell surface receptors on hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily and have been shown to mediate the removal of antibody-coated pathogens by phagocytosis of immune complexes and the lysis of corresponding antibody-coated red blood cells and other various cellular targets (e.g., tumor cells) by antibody-dependent cell-mediated cytotoxicity (ADCC) (see, e.g., Van de Winkel, j.g., and Anderson, c.l., j.leukc.biol.49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin isotypes: the Fc receptor of IgG antibodies is called Fc γ R. Fc receptor binding is described, for example, in: ravech, J.V. and Kinet, J.P., Annu. revision, Immunol.9(1991) 457-; capel, P.J. et al, Immunomethods 4(1994) 25-34; de Haas, M. et al, J.Lab.Clin.Med.126(1995) 330-; and Gessner, J.E., et al, Ann.Hematol.76(1998) 231-.

Cross-linking of IgG antibody (fcyr) Fc region receptors triggers a variety of effector functions, including phagocytosis, antibody-dependent cellular cytotoxicity, release of inflammatory mediators, and immune complex clearance and modulation of antibody production. Three classes of Fc γ rs have been identified in humans, including:

Fc γ RI (CD64) binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils. Modifications in at least one of amino acid residues E233-G236, P238, D265, N297, a327 and P329 (numbering according to EU index of Kabat) in the IgG of the Fc region reduced binding to Fc γ RI. The IgG2 residues at position 233-³Double, and abolished the response of human monocytes to antibody-sensitized erythrocytes (Armour, K.L. et al, Eur.J. Immunol.29(1999) 2613-2624).

Fc γ RII (CD32) binds complex IgG with moderate to low affinity and is widely expressed. The receptors can be divided into two subtypes, Fc γ RIIA and Fc γ RIIB. Fc γ RIIA is present in many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. Fc γ RIIB appears to play a role in the inhibition process and is present in B cells, macrophages, as well as mast cells and eosinophils. On B cells, it appears to act to inhibit further immunoglobulin production and isotype switching to, for example, IgE class. On macrophages, Fc γ RIIB is used to inhibit phagocytosis mediated by Fc γ RIIA. On eosinophils and mast cells, type B may help to inhibit activation of these cells by binding of IgE to its individual receptor. Reduced binding of e.g. an antibody (comprising a mutated IgG Fc region at least one of amino acid residues E233-G236, P238, D265, N297, a327, P329, D270, Q295, a327, R292 and K414 (numbering according to EU index of Kabat)) to Fc γ RIIA was found.

Fc γ RIII (CD16) binds IgG with moderate to low affinity and includes both types. Fc γ RIIIA is present on NK cells, macrophages, eosinophils, and some monocytes and T cells, and mediates ADCC. Fc γ RIIIB is expressed at high levels on neutrophils. Reduced binding to Fc γ RIIIA was found, for example, in antibodies comprising mutated IgG Fc regions at least one of amino acid residues E233-G236, P238, D265, N297, a327, P329, D270, Q295, a327, S239, E269, E293, Y296, V303, a327, K338 and D376 (numbering according to the EU index of Kabat).

Shields, r.l. et al (j.biol.chem.276(2001)6591-6604) describe the location of the binding site on human IgG1 to Fc receptors, the above mentioned mutation sites and methods for measuring binding to Fc γ RI and Fc γ RIIA.

The term "ADCC" or "antibody dependent cellular cytotoxicity" is a function mediated by Fc receptor binding and refers to the lysis of target cells by an antibody as reported herein in the presence of effector cells. The ability of an antibody to induce an initial step in mediating ADCC is investigated by measuring the binding of the antibody to cells expressing Fc γ receptors, such as cells recombinantly expressing Fc γ RI and/or Fc γ RIIA or NK cells (essentially expressing Fc γ RIIIA). Specifically, binding to Fc γ R on NK cells was measured.

An "activating Fc receptor" is an Fc receptor that, upon engagement of the Fc region of an antibody, causes a signaling event that stimulates receptor-bearing cells to perform effector functions. Activating Fc receptors include Fc γ RIIIa (CD16a), Fc γ RI (CD64), Fc γ RIIa (CD32), and Fc α RI (CD 89). A particular activating Fc receptor is human Fc γ RIIIa (see UniProt accession No. P08637, version 141).

The "tumor necrosis factor receptor superfamily" or "TNF receptor superfamily" currently consists of 27 receptors. It is a group of cytokine receptors characterized by the ability to bind Tumor Necrosis Factor (TNF) through the extracellular cysteine-rich domain (CRD). These pseudo-repeats are defined by intra-chain disulfide bonds formed by highly conserved cysteine residues within the acceptor chain. All TNF was homologous to prototype TNF- α, except for Nerve Growth Factor (NGF). Most TNF receptors form trimeric complexes in plasma membranes in their active form. Thus, most TNF receptors contain a transmembrane domain (TMD). Many of these receptors also contain intracellular Death Domains (DD) that recruit proteins that interact with caspases upon ligand binding, thereby initiating the exogenous pathway of caspase activation. Other TNF superfamily receptors lacking a death domain bind to TNF receptor associated factors and activate intracellular signaling pathways, leading to proliferation or differentiation. These receptors may also initiate apoptosis, but they act via an indirect mechanism. In addition to modulating apoptosis, a variety of TNF superfamily receptors are involved in modulating immune cell functions such as B cell homeostasis and activation, natural killer cell activation, and T cell co-stimulation. A variety of other agents modulate specific cell type responses such as hair follicle development and osteoclast development. Members of the TNF receptor superfamily include: tumor necrosis factor receptor 1(1A) (TNFRSF1, CD 120), tumor necrosis factor receptor 2(1B) (TNFRSF1, CD 120), lymphotoxin beta receptor (LTBR, CD), OX (TNFRSF, CD134), CD (Bp), Fas receptor (Apo-1, CD, FAS), decoy receptor 3(TR, M, TNFRSF 6), CD (S152, Tp), CD (Ki-1, TNFRSF), 4-1BB (CD137, TNFRSF), DR (TRAILR, Apo-2, CD261, TNFRSF 10), DR (TRAILR, CD262, TNFRSF 10), decoy receptor 1(TRAILR, CD263, TNFRSF 10), decoy receptor 2(TRAILR, CD264, TNFRSF 10), RANK (CD, TNFRSF 11), osteoprotegerin (OCIF, TR, TNFRSF 11), TNFRSF11, TNFRSF 271, CD266, HVRSF 12, TNFRSF13, TNFRSF17), glucocorticoid-induced TNFR-related (GITR, AITR, CD357, TNFRSF18), TROY (TNFRSF19), DR6(CD358, TNFRSF21), DR3(Apo-3, TRAMP, WS-1, TNFRSF25), and the epiblastic aplasia a2 receptor (xedr, EDA 2R).

Multiple members of the Tumor Necrosis Factor Receptor (TNFR) family maintain T cell responses after initial T cell activation. The term "co-stimulatory TNF receptor family members" or "co-stimulatory TNF family receptors" refers to a subset of TNF receptor family members that are capable of synergistically stimulating T cell proliferation and cytokine production. Unless otherwise indicated, the term refers to any native TNF family receptor from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). In a specific embodiment of the invention, the co-stimulatory TNF receptor family member is selected from the group consisting of: OX40(CD134), 4-1BB (CD137), CD27, HVEM (CD270), CD30 and GITR, all of which have a co-stimulatory effect on T cells. More specifically, the co-stimulatory TNF receptor family member is 4-1 BB.

The term "4-1 BB" as used herein, unless otherwise indicated, refers to any native 4-1BB from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term includes "full-length" unprocessed 4-1BB, as well as any form of 4-1BB produced by processing in a cell. The term also encompasses naturally occurring variants of 4-1BB, such as splice variants or allelic variants. The amino acid sequence of exemplary human 4-1BB is shown in SEQ ID NO:97(Uniprot accession No. Q07011), the amino acid sequence of exemplary murine 4-1BB is shown in SEQ ID NO:98(Uniprot accession No. P20334), and the amino acid sequence of exemplary cynomolgus monkey 4-1BB (from macaque) is shown in SEQ ID NO:99(Uniprot accession No. F6W5G 6).

The terms "anti-4-1 BB antibody," anti-4-1 BB, "" 4-1BB antibody, and "antibody that specifically binds to 4-1 BB" refer to an antibody that is capable of binding 4-1BB with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent targeting 4-1 BB. In one embodiment, anti-4-1 BB antibody is detected, for example, by Radioimmunoassay (RIA) or flow cytometry (FACS)The degree of binding of the antibody to an unrelated, non-4-1 BB protein is less than about 10% of the degree of binding of the antibody to 4-1 BB. In certain embodiments, the dissociation constant (K) of an antibody that binds 4-1BB_D) Is ≤ 1 μ M, ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-6M or less, e.g. 10^-68M to 10^-13M, e.g. 10^-8M to 10^-10M). Specifically, the anti-4-1 BB antibody is clone 20H4.9 disclosed in U.S. Pat. No. 7,288,638.

The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G)₄S)_n、(SG₄)_nOr G₄(SG₄)_nA peptide linker, wherein "n" is typically a number between 1 and 10, typically between 2 and 4, in particular 2, i.e. a peptide selected from the group consisting of: GGGGS (SEQ ID NO:100), GGGGSGGGGS (SEQ ID NO:101), SGGGGSGGGG (SEQ ID NO:102), and GGGGSGGGGSGGGG (SEQ ID NO:103), but also includes the following sequences: GSPGSSSSGS (SEQ ID NO:104), (G4S) ₃(SEQ ID NO:105)、(G4S)₄(SEQ ID NO:106), GSGSGSGS (SEQ ID NO:107), GSGSGNGS (SEQ ID NO:108), GGSGSGSGSG (SEQ ID NO:109), GGSGSG (SEQ ID NO:110), GGSG (SEQ ID NO:111), GGSGNGSG (SEQ ID NO:112), GGNGSGSG (SEQ ID NO:113) and GGNGSG (SEQ ID NO: 114). Peptide linkers of particular interest are (G4S) (SEQ ID NO:100), (G)₄S)₂Or GGGGSGGGGS (SEQ ID NO:101), (G4S)₃(SEQ ID NO:105) and (G4S)₄(SEQ ID NO:106), and more specifically (G)₄S)₂Or GGGGSGGGGS (SEQ ID NO: 101).

The term "amino acid" as used in this application denotes the group of naturally occurring carboxy alpha-amino acids comprising: alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

By "fusion" or "linked" is meant that the components (e.g., the heavy chains of the antibody and Fab fragments) are linked by peptide bonds, either directly or via one or more peptide linkers.

"percent amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in a reference polypeptide sequence, after aligning the candidate sequence with the reference polypeptide sequence and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and for purposes of alignment, without regard to any conservative substitutions as part of the sequence identity. Alignments to determine percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithms required to achieve maximum alignment over the full length of the sequences being compared. Alternatively, a percent identity value can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted with the user document to u.s.copy Office, Washington d.c.,20559, where it was registered with us copyright registration number TXU510087 and as described in WO 2001/007611.

In certain embodiments, amino acid sequence variants of the antigen binding molecules comprising TNF ligand trimers provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antigen binding molecule comprising a trimer of TNF ligands. Amino acid sequence variants of the antigen-binding molecules comprising TNF ligand trimers can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen binding. Sites of interest for substitution mutagenesis include HVRs and Frameworks (FRs). Conservative substitutions are provided below the head "preferred substitutions" in table a and are described further below with reference to amino acid side chain classes (1) to (6). Amino acid substitutions can be introduced into the molecule of interest and the product screened for a desired activity (e.g., retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

TABLE A

Amino acids can be grouped according to common side chain properties:

(1) hydrophobicity; norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln;

(3) acidity: asp and Glu;

(4) alkalinity: his, Lys, Arg;

(5) residues that influence chain orientation: gly, Pro;

(6) aromatic: trp, Tyr, Phe.

Non-conservative substitutions will require the exchange of a member of one of these classes for another.

The term "amino acid sequence variant" includes substantial variants in which there is an amino acid substitution in one or more hypervariable region residues of a parent antigen-binding molecule (e.g., a humanized or human antibody). Typically, one or more of the resulting variants selected for further study will be altered (e.g., improved) in certain biological properties (e.g., increased affinity, decreased immunogenicity) and/or will substantially retain certain biological properties of the parent antigen-binding molecule relative to the parent antigen-binding molecule. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and variant antigen binding molecules are displayed on phage and screened for a particular biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such alterations do not substantially reduce the antigen-binding ability of the antigen-binding molecule. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. A method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is referred to as "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science,244: 1081-1085. In this method, a residue or set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether antibody interaction with an antigen is affected. Additional substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antigen binding molecule complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues that are candidates for substitution may be targeted or eliminated. Variants can be screened to determine if they possess the desired properties.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include a bispecific antigen binding molecule of the invention with an N-terminal methionyl residue. Other insertional variants of the molecule include fusions to the N-terminus or C-terminus of the polypeptide that increases the serum half-life of the bispecific antigen binding molecule.

In certain embodiments, the bispecific antigen binding molecules provided herein are altered to increase or decrease the degree of antibody glycosylation. Glycosylated variants of the molecule may conveniently be obtained by altering the amino acid sequence such that one or more glycosylation sites are created or removed. When an antigen binding molecule comprising a TNF ligand trimer comprises an Fc region, the carbohydrate attached thereto can be altered. Natural antibodies produced by mammalian cells typically comprise bi-antennary oligosaccharides with a branched chain, typically attached through an N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some embodiments, the oligosaccharides in an antigen binding molecule comprising a trimer of a TNF family ligand can be modified to produce variants with certain improved properties. In one aspect, variants of the bispecific antigen binding molecules or antibodies of the invention are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. Such fucosylated variants may have improved ADCC function, see, e.g., U.S. patent publication No. US 2003/0157108(Presta, L.) or US 2004/0093621(Kyowa Hakko Kogyo co., Ltd.). In another aspect, the bispecific antigen binding molecules or antibodies of the invention are provided with a variant of bisected oligosaccharides, for example wherein the biantennary oligosaccharides attached to the Fc region are bisected by GlcNAc. Such variants may have reduced fucosylation and/or improved ADCC function, see for example WO 2003/011878(Jean-Mairet et al); U.S. Pat. No. 6,602,684(Umana et al); and US 2005/0123546(Umana et al). Also provided are variants having at least one galactose residue in an oligosaccharide attached to an Fc region. Such antibody variants may have improved CDC function and are described, for example, in WO 1997/30087(Patel et al); WO 1998/58964(Raju, S.); and WO 1999/22764(Raju, S.).

In certain aspects, it may be desirable to produce cysteine engineered variants of the bispecific antigen binding molecules of the invention, e.g., "thiomabs," in which one or more residues of the molecule are substituted with a cysteine residue. In particular aspects, the substituted residue is present at an accessible site on the molecule. By replacing those residues with cysteine, the reactive thiol group is thereby localized to an accessible site of the antibody and can be used to conjugate the antibody to other moieties, such as a drug moiety or linker-drug moiety, to produce an immunoconjugate. In certain aspects, any one or more of the following residues may be substituted with cysteine: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antigen binding molecules can be formed as described, for example, in U.S. patent No. 7,521,541.

The term "nucleic acid molecule" or "polynucleotide" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, in particular a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (a), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and a phosphate group. Generally, nucleic acid molecules are described by the sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is usually expressed from 5 'to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cdna) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mrna)), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-and double-stranded forms. In addition, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and/or in vivo (e.g., in a host or patient). Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoding molecule so that the mRNA can be injected into a subject to produce in vivo antibodies (see, e.g., Stadler et al, Nature Medicine 2017, published on 2017 at 12.6.7, doi:10.1038/nm.4356 or EP 2101823B 1).

An "isolated" polynucleotide refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

By "isolated polynucleotide encoding a bispecific antigen binding molecule" is meant one or more nucleic acid molecules encoding the heavy and light chains of an antibody (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, as well as such nucleic acid molecules present at one or more locations in a host cell.

The term "expression cassette" refers to a polynucleotide, generated recombinantly or synthetically, with a series of specific nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, among other sequences, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassettes of the invention comprise a polynucleotide sequence encoding a bispecific antigen binding molecule of the invention or a fragment thereof.

The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule for introducing a particular gene into a target cell with which it is operably associated and directing the expression of the gene. The term includes vectors which are self-replicating nucleic acid structures, as well as vectors which integrate into the genome of a host cell into which they have been introduced. The expression vector of the present invention comprises an expression cassette. Expression vectors allow for the transcription of a large number of stable mrnas. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by cellular transcription and/or translation machinery. In one embodiment, the expression vector of the invention comprises an expression cassette comprising a polynucleotide sequence encoding the bispecific antigen binding molecule of the invention or a fragment thereof.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include a primary transformed cell and progeny derived from the primary transformed cell, regardless of the number of passages. Progeny may not be completely identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The host cell is any type of cellular system that can be used to produce the bispecific antigen binding molecules of the invention. Host cells include cultured cells, for example, cultured mammalian cells such as CHO cells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, per.c6 cells or hybridoma cells, yeast cells, insect cells and plant cells, as well as cells included in transgenic animals, transgenic plants or cultured plant or animal tissues, to name a few.

An "effective amount" of an agent is that amount necessary to produce a physiological change in the cell or tissue to which it is administered.

A "therapeutically effective amount" of an agent (e.g., a pharmaceutical composition) is an amount effective to achieve the desired therapeutic or prophylactic result at the necessary dosage and for the period of time. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, the individual or subject is a human.

The term "pharmaceutical composition" refers to a formulation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that is free of additional components that have unacceptable toxicity to the subject to which the formulation is to be administered.

"pharmaceutically acceptable excipient" refers to an ingredient of a pharmaceutical composition other than an active ingredient that is not toxic to a subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, or preservatives.

The term "package insert" is used to refer to instructions typically included in commercial packaging for therapeutic products that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings concerning the use of such therapeutic products.

As used herein, "treatment" (and grammatical variations thereof, such as "treatment" or "treating") refers to a clinical intervention that attempts to alter the natural course of the treated individual, and may be for the purpose of prevention or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the molecules of the invention are used to delay the progression of a disease or to slow the progression of a disease.

The term "cancer" as used herein refers to a proliferative disease, such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer (stomachs), stomach cancer (gastrostatic cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, carcinoma of the esophagus, carcinoma of the small intestine, carcinoma of the endocrine system, carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, prostate cancer, carcinoma of the bladder, carcinoma of the kidney or ureter, carcinoma of the renal cell, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, cholangiocarcinoma, neoplasms of the Central Nervous System (CNS), rachis tumor, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and ewing's sarcoma, including refractory forms of any of the above cancers, or combinations of one or more of the above cancers.

Bispecific antigen binding molecules of the invention

The present invention relates to novel bispecific agonistic 4-1BB antibodies with particularly advantageous properties, such as producibility, stability, binding affinity, biological activity, targeting efficiency and reduced toxicity. They are also characterized by their specific structural features such as proximity of two Fab fragments capable of specifically binding to 4-1BB and the target cell antigen, respectively, bivalent binding to 4-1BB and monovalent binding to the target cell antigen making these bispecific antigen binding molecules very effective and not affecting safety.

Exemplary bispecific antigen binding molecules

(a) a first Fab fragment capable of specifically binding to 4-1 BB;

(c) a third Fab fragment capable of specifically binding to 4-1 BB; and

(a) a first Fab fragment capable of specifically binding to 4-1 BB;

(c) a third Fab fragment capable of specifically binding to 4-1 BB; and

(d) an Fc domain consisting of a first subunit and a second subunit capable of stable association; wherein the second Fab fragment (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab fragment (a), which in turn is fused at its C-terminus to the N-terminus of the first Fc domain subunit, and the third Fab fragment (C) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit, and wherein in the second Fab fragment capable of specific binding to a target cell antigen, the variable regions VL and VH of the Fab light chain and Fab heavy chain are replaced with each other.

Specifically, a Fab fragment capable of specifically binding to 4-1BB comprises: heavy chain variable region (V)_H4-1BB) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and light chain variable region (V) _L4-1BB) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

In a particular aspect, there is provided a bispecific antigen binding molecule as described herein, wherein the bispecific antigen binding molecule provides bivalent binding to 4-1BB and monovalent binding to a target cell antigen. More specifically, the bispecific antigen binding molecules provide bivalent binding to 4-1BB and monovalent binding to Tumor Associated Antigens (TAAs).

The Fab fragment may be fused to the Fc domain directly or via a peptide linker comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art and described herein. Suitable non-immunogenic peptide linkers include, for example, (G4S) n, (SG4) n, (G4S) n or G4(SG4) n peptide linkers. "n" is typically an integer from 1 to 10, typically from 2 to 4. Adapted for linking a first Fab fragmentAn exemplary peptide linker of the Fab heavy chain of the fragment and the second Fab fragment comprises the sequences (D) - (G)₄S)₂. Another suitable such linker comprises the sequence (G)₄S)₄. Additionally, the linker may comprise (a part of) an immunoglobulin hinge region. In particular, in case the Fab molecule is fused to the N-terminus of the Fc domain subunit, the fusion may be via an immunoglobulin hinge region or a portion thereof, with or without additional peptide linkers.

In one aspect, there is provided a bispecific antigen binding molecule as described above, wherein the first and third Fab fragments capable of specific binding to 4-1bb, (a) and (c) are the same. In a particular aspect, the invention provides a bispecific antigen binding molecule as described above, wherein the first and third Fab fragments capable of specific binding to 4-1BB each comprise: heavy chain variable region (V)_H4-1BB) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and light chain variable region (V)_L4-1BB) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

Accordingly, in one aspect, there is provided a bispecific antigen binding molecule comprising:

(a) a first Fab fragment capable of specifically binding to 4-1 BB;

(c) a third Fab fragment capable of specifically binding to 4-1 BB; and

(d) An Fc domain consisting of a first subunit and a second subunit capable of stable association; wherein

The second Fab fragment (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab fragment (a), which in turn is fused at its C-terminus to the N-terminus of the first Fc domain subunit, and the third Fab fragment (C) is fused at the C-terminus of the Fab heavy chain to the second Fc junctionThe N-terminus of the domain subunit, and wherein in a second Fab fragment capable of specific binding to a target cell antigen, (i) the variable regions VL and VH of the Fab light chain and Fab heavy chain are replaced with each other, or (ii) the constant regions CL and CH1 of the Fab light chain and Fab heavy chain are replaced with each other, and wherein the first Fab fragment and the third Fab fragment each comprise: heavy chain variable region (V)_H4-1BB) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and light chain variable region (V)_L4-1BB) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

In a particular aspect, a bispecific antigen binding molecule comprises a first Fab fragment and a third Fab fragment capable of specifically binding to 4-1BB, each comprising: heavy chain variable region (V) _H4-1BB) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 7; and light chain variable region (V)_L4-1BB) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 8. More specifically, the first and third Fab fragments capable of specifically binding to 4-1BB each comprise: heavy chain variable region (V)_H4-1BB) comprising the amino acid sequence of SEQ ID NO 7; and light chain variable region (V)_L4-1BB) comprising the amino acid sequence of SEQ ID NO. 8.

In one aspect, the bispecific antigen binding molecule comprises one polypeptide comprising the amino acid sequence of SEQ ID NO:65 (Fc pore heavy chain) and two polypeptides comprising the amino acid sequence of SEQ ID NO:67 (light chain).

The bispecific antigen binding molecules of the invention are also characterized by a Fab fragment capable of specifically binding to a target cell antigen. Thus, bispecific antigen binding molecules have the advantage over conventional antibodies capable of specifically binding to 4-1BB that they selectively induce a costimulatory T cell response on target cells (typically cancer cells). In one aspect, the target cell antigen is selected from the group consisting of: fibroblast Activation Protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, CD33, and PD-L1. In one aspect, the target cell antigen is selected from the group consisting of: fibroblast Activation Protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, and CD 33. Specifically, the target cell antigen is selected from Fibroblast Activation Protein (FAP), carcinoembryonic antigen (CEA), and CD 19. In a particular aspect, the target cell antigen is selected from Fibroblast Activation Protein (FAP) and carcinoembryonic antigen (CEA). More specifically, the target cell antigen is FAP. Alternatively, the target cell antigen is CEA. In another particular aspect, the target cell antigen is CD 19. In a further aspect, the target cell antigen is PD-L1.

Bispecific antigen binding molecules wherein the target cell antigen is FAP

In a particular aspect, the target cell antigen is Fibroblast Activation Protein (FAP). FAP incorporation has been described in part in WO 2012/02006, which is incorporated herein by reference in its entirety. The FAP binding moieties of particular interest are described below.

In one aspect, the present invention provides a bispecific antigen binding molecule, wherein a Fab fragment capable of specific binding to Fibroblast Activation Protein (FAP) comprises:

(b) Heavy chain variable region (V)_HFAP) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:15, (ii) CDR-H2 comprising the amino group of SEQ ID NO:16(ii) a sequence, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and light chain variable region (V) _LFAP) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:18, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:19, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In particular, there is provided a bispecific antigen binding molecule, wherein a Fab fragment capable of specifically binding to FAP comprises: heavy chain variable region (V)_HFAP) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and light chain variable region (V)_LFAP) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:13, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14.

In another aspect, a Fab fragment capable of specifically binding to FAP comprises: heavy chain variable region (V)_HFAP) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:15, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:16, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and light chain variable region (V)_LFAP) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:18, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:19, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

In particular, there is provided a bispecific antigen binding molecule, wherein a Fab fragment capable of specific binding to Fibroblast Activation Protein (FAP) comprises:

(a) heavy chain variable region (V)_HFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 21; and light chain variable region (V)_LFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 22; or

(b) Heavy chain variable region (V)_HFAP) of the packetComprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 23; and light chain variable region (V)_LFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 24.

In particular, a Fab fragment capable of specifically binding to FAP comprises: heavy chain variable region (V)_HFAP) comprising the amino acid sequence of SEQ ID NO:21, and a light chain variable region (V)_LFAP) comprising the amino acid sequence of SEQ ID NO 22; or heavy chain variable region (V)_HFAP) comprising the amino acid sequence of SEQ ID NO:23, and a light chain variable region (V) _LFAP) comprising the amino acid sequence of SEQ ID NO 24. More specifically, a Fab fragment capable of specifically binding to FAP comprises: heavy chain variable region (V)_HFAP) comprising the amino acid sequence of SEQ ID NO 21; and light chain variable region (V)_LFAP) comprising the amino acid sequence of SEQ ID NO 22.

In another aspect, a bispecific antigen binding molecule is provided, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:21 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 22.

In a further aspect, there is provided a bispecific antigen binding molecule, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:23 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 24.

In a particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 66; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 68. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 66, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 68.

Bispecific antigen binding molecules wherein the target cell antigen is CEA

In a particular aspect, the target cell antigen is carcinoembryonic antigen (CEA). CEA binding moieties have been described, for example, in WO 92/01059, WO 2007/071422, WO 2016/075278A2 or WO2007/071426, which are incorporated herein by reference in their entirety. CEA binding moieties of particular interest are described below.

In one aspect, the present invention provides a bispecific antigen binding molecule wherein a Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises:

(c) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:42, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and light chain variable region (V) _LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:45, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; or

(e) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:115, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:116, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 117; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:118, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:119, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 120; or

(f) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:123, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:124 or SEQ ID NO:125, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 126; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:127 or SEQ ID NO:128, (v)(vii) CDR-L2 comprising the amino acid sequence of SEQ ID NO:129 or SEQ ID NO:130 or SEQ ID NO:131, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 132.

In particular, a bispecific antigen binding molecule is provided, wherein a Fab fragment capable of specific binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and light chain variable region (V)_LCEA) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30.

In particular, a bispecific antigen binding molecule is provided, wherein a Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises: (a) heavy chain variable region (V) _HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 31; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 32. In one aspect, a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA) comprises: (a) heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 31; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO:32 (antibody A5B 7).

In one aspect, a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA) comprises: humanized heavy chain variable region (V)_HCEA) based on the human receptor framework IGHV3-23-02 comprising the amino acid sequence of SEQ ID NO 153; and humanized light chain variable region (V)_LCEA) based on the human receptor framework IGKV3-11 comprising the amino acid sequence of SEQ ID No. 165.

In a particular aspect, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO 115(ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:116, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 117; and light chain variable region (V) _LCEA) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:118, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:119, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 120.

In one aspect, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 121; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 122. Specifically, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO. 121; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO:122 (antibody A5H1EL 1D). In another aspect, a Fab fragment can comprise an antibody having a higher affinity for CEA than A5H1EL1D, wherein A5H1EL1D comprises the same framework regions but with mutations in the CDR regions. Thus, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising an amino acid sequence at least about 95% identical to the amino acid sequence of SEQ ID NO. 121; and light chain variable region (V) _LCEA) comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID No. 122.

In another aspect, a Fab fragment capable of specifically binding to FAP comprises: heavy chain variable region (V)_HFAP) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:33, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:34, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35; and light chain variable region (V)_LFAP) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:36, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:37, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38.

In one aspect, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 39; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 40. In one aspect, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V) _HCEA) comprising the amino acid sequence of SEQ ID NO 39; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO:40 (antibody MFE 23).

In particular, a bispecific antigen binding molecule is provided, wherein a Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises humanized heavy and light chain variable domains. In one aspect, a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA) comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137 or SEQ ID NO 138; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 139, 140, 141, 142, 143 or 144.

In one aspect, a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA) comprises:

(a) heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 133 and the light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO. 143; or

(b) Heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:137, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO. 143; or

(c) Heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:134, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO. 143; or

(d) Heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:138, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO: 142; or

(e) Heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:137, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO: 142; or

(f) Heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO:135, and a light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO: 142; or

(g) Heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 133 and the light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO: 142.

In a further aspect, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:42, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and light chain variable region (V) _LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:45, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46.

In one aspect, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 47; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 48. Specifically, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 47; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO:48 (antibody T84.66-LCHA).

In a further aspect, canThe Fab fragment that binds specifically to CEA comprises: heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:50, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 51; and light chain variable region (V) _LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:52, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:53, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54.

In one aspect, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 56. Specifically, a Fab fragment capable of specifically binding to CEA comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO: 55; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO:56 (antibody CH1A1A 98/99/2F 1).

In one aspect, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO: 31; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 32; or an antigen-binding domain capable of specifically binding to FAP comprising: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO: 39; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 40.

In another aspect, the antigen binding domain capable of specifically binding to CEA comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 48; or an antigen-binding domain capable of specifically binding to FAP comprising: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO: 55; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 56.

In another aspect, a bispecific antigen binding molecule is provided, wherein

(i) The first and third Fab fragments capable of specifically binding to 4-1BB comprise: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO 8; and is

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:31 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 32.

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:39 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 40.

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:47 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 48.

In another aspect, a bispecific antigen binding molecule is provided, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:55 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 56.

In another aspect, a bispecific antigen binding molecule is provided, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: the heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:121 and the light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 122.

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:133 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 143.

In another aspect, a bispecific antigen binding molecule is provided, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:137 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 143.

In another aspect, a bispecific antigen binding molecule is provided, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:134 and a light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 143.

In yet another aspect, a bispecific antigen binding molecule is provided, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: the heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:138 and the light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 142.

In another aspect, a bispecific antigen binding molecule is provided, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: the heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:137 and the light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 142.

In another aspect, a bispecific antigen binding molecule is provided, wherein

(ii) A second Fab fragment capable of specifically binding to FAP comprises: the heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:135 and the light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 142.

(ii) A second Fab fragment capable of specifically binding to FAP comprises: the heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:133 and the light chain variable region VH comprising the amino acid sequence of SEQ ID NO: 142.

In a particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 76; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 77. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 76, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 77.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 78; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 79. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 78, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 79.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 80; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 81. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 80, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 81.

In yet another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 82; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 83. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 82, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 83.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 173; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 174. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 173, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 174.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 179; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 180. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 179, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 180.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 181; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 182. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 181, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 182.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 183; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 184. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 183, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 184.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 185; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 186. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 185, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 186.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO. 187; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO. 188. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 187, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 188.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 189; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 190. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 189, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 190.

In another particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 191; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 192. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 191, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 192.

Bispecific antigen binding molecules wherein the target cell antigen is CD19

In a particular aspect, the target cell antigen is CD 19. CD19 binding moieties have been described, for example, in WO 2016/075278 a1, which is incorporated herein by reference in its entirety. CD19 binding moieties of particular interest are described below.

In one aspect, the present invention provides a bispecific antigen binding molecule, wherein a Fab fragment capable of specific binding to CD19 comprises:

Specifically, a Fab fragment capable of specifically binding to CD19 comprises: heavy chain variable region (V)_HCD19) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 63; and light chain variable region (V) _LCD19) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 64. More specifically, a Fab fragment capable of specifically binding to CD19 comprises: heavy chain variable region (V)_HCD19) comprising the amino acid sequence of SEQ ID NO:63, and a light chain variable region (V)_LCD19) comprising the amino acid sequence of SEQ ID NO: 64.

In a particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 84; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 85. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 84, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 85.

Bispecific antigen binding molecules wherein the target cell antigen is PD-L1

In a particular aspect, the target cell antigen is PD-L1. CD19 binding moieties have been described, for example, in WO 2010/077634, which is incorporated herein by reference in its entirety. PD-L1 binding moieties of particular interest are described below.

In one aspect, the present invention provides a bispecific antigen binding molecule, wherein a Fab fragment capable of specific binding to PD-L1 comprises:

heavy chain variable region (V)_HPD-L1) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:145, (ii) CDR-H2 comprising SE(ii) the amino acid sequence of Q ID NO:146, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 147; and light chain variable region (V)_LPD-L1) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:148, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:149, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 150.

Specifically, a Fab fragment capable of specifically binding to PD-L1 comprises: heavy chain variable region (V)_HPD-L1) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 152; and light chain variable region (V) _LPD-L1) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 153. More specifically, a Fab fragment capable of specifically binding to PD-L1 comprises: heavy chain variable region (V)_HPD-L1) comprising the amino acid sequence of SEQ ID NO:152, and a light chain variable region (V)_LPD-L1) comprising the amino acid sequence of SEQ ID NO: 153.

(ii) A second Fab fragment capable of specifically binding to CD19 comprises: a heavy chain variable region VH comprising the amino acid sequence of SEQ ID NO:152 and a light chain variable region VL comprising the amino acid sequence of SEQ ID NO: 153.

In a particular aspect, a bispecific antigen binding molecule comprises: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO 193; two polypeptides that are at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 194. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 193, both polypeptide sequences of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 194.

Also disclosed is an antigen binding molecule (1+1 form) having monovalent binding to 4-1 BB. Accordingly, there is provided a bispecific antigen binding molecule comprising: a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 65; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO: 195; a polypeptide that is at least 95%, 96%, 97%, 98% or 99% identical to the sequence of SEQ ID NO. 67; and a polypeptide that is at least 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 194. In yet another embodiment, the bispecific antigen binding molecule comprises the polypeptide sequence of SEQ ID NO 65, the polypeptide sequence of SEQ ID NO 195, the polypeptide sequence of SEQ ID NO 67 and the polypeptide sequence of SEQ ID NO 194.

Fc domain modifications that reduce Fc receptor binding and/or effector function

The bispecific antigen binding molecules of the present invention further comprise an Fc domain consisting of a first subunit and a second subunit capable of stable binding.

In certain aspects, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising amino acid modifications (e.g., substitutions) at one or more amino acid positions.

The Fc domain confers advantageous pharmacokinetic properties to the bispecific antibodies of the invention, including a long serum half-life that contributes to good accumulation in the target tissue and a favorable tissue-to-blood partition ratio. At the same time, however, it may result in the bispecific antibodies of the invention undesirably targeting Fc receptor expressing cells rather than the preferred antigen carrying cells. Thus, in particular embodiments, the Fc domain of the bispecific antibodies of the invention exhibits reduced binding affinity to an Fc receptor and/or reduced effector function compared to a native IgG Fc domain, in particular an IgG1Fc domain or an IgG4 Fc domain. More specifically, the Fc domain is an IgG1Fc domain.

In one such aspect, the Fc domain (or bispecific antigen binding molecule of the invention comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity for an Fc receptor as compared to a native IgG1Fc domain (or bispecific antigen binding molecule of the invention comprising a native IgG1Fc domain); and/or the Fc domain (or bispecific antigen binding molecule of the invention comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of effector function compared to a native IgG1Fc domain (or bispecific antigen binding molecule of the invention comprising a native IgG1Fc domain). In one aspect, the Fc domain (or bispecific antigen binding molecule of the invention comprising the Fc domain) does not significantly bind to an Fc receptor and/or induce effector function. In a particular aspect, the Fc receptor is an fey receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activated Fc receptor. In a particular aspect, the Fc receptor is an activated human Fc γ receptor, more particularly human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most particularly human Fc γ RIIIa. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a particular aspect, the Fc receptor is an inhibitory human Fc γ receptor, more particularly human Fc γ RIIB. In one aspect, the effector function is one or more of CDC, ADCC, ADCP and cytokine secretion. In a particular aspect, the effector function is ADCC. In one aspect, the Fc domain exhibits substantially similar binding affinity to a neonatal Fc receptor (FcRn) as compared to a native IgG1Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or bispecific antigen binding molecule of the invention comprising said Fc domain) exhibits greater than about 70%, specifically greater than about 80%, more specifically greater than about 90% of the binding affinity of the native IgG1Fc domain (or bispecific antigen binding molecule of the invention comprising a native IgG1Fc domain) for FcRn.

In a particular aspect, the Fc domain is engineered to have reduced binding affinity to an Fc receptor and/or reduced effector function as compared to a non-engineered Fc domain. In a particular aspect, the Fc domain of the bispecific antigen binding molecules of the invention comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to an Fc receptor and/or effector function. Typically, the same amino acid mutation or mutations are present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor. In another aspect, the amino acid mutation reduces the binding affinity of the Fc domain to an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold. In one aspect, bispecific antigen binding molecules of the invention comprising an engineered Fc domain exhibit less than 20%, specifically less than 10%, more specifically less than 5% of the binding affinity to an Fc receptor compared to bispecific antibodies of the invention comprising a non-engineered Fc domain. In a particular aspect, the Fc receptor is an fey receptor. In other aspects, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a particular aspect, the Fc receptor is an inhibitory human Fc γ receptor, more particularly human Fc γ RIIB. In some aspects, the Fc receptor is an activated Fc receptor. In a particular aspect, the Fc receptor is an activated human Fc γ receptor, more particularly human Fc γ RIIIa, Fc γ RI or Fc γ RIIa, most particularly human Fc γ RIIIa. Preferably, the binding to each of these receptors is reduced. In some aspects, the binding affinity to the complementary component, the specific binding affinity to C1q is also reduced. In one aspect, the binding affinity for neonatal Fc receptor (FcRn) is not reduced. Substantially similar binding to FcRn, i.e., retention of the binding affinity of the Fc domain for the receptor, is achieved when the Fc domain (or the bispecific antigen binding molecule of the invention comprising an Fc domain) exhibits greater than about 70% of the binding affinity of the unengineered form of the Fc domain (or the bispecific antigen binding molecule of the invention comprising an unengineered form of the Fc domain) for FcRn. The Fc domain or bispecific antigen binding molecule of the invention comprising the Fc domain may exhibit greater than about 80% or even greater than about 90% of such affinity. In certain embodiments, the Fc domain of the bispecific antigen binding molecules of the invention is engineered to have reduced effector function compared to a non-engineered Fc domain. Reduced effector function may include, but is not limited to, one or more of the following: reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell mediated cytotoxicity (ADCC), reduced Antibody Dependent Cellular Phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex mediated antigen uptake by antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

Antibodies with reduced effector function include those with substitutions of one or more of residues 238, 265, 269, 270, 297, 327 and 329 of the Fc region (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acids 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are substituted with alanine (U.S. Pat. No. 7,332,581). Certain antibody variants with improved or reduced binding to FcR are described. (e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, and Shields, R.L. et al, J.biol.chem.276(2001) 6591-.

In one aspect of the invention, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331 and P329. In some aspects, the Fc domain comprises the amino acid substitutions L234A and L235A ("LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more particular aspect, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and comprises a further amino acid substitution selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a more specific embodiment, the Fc domain comprises the amino acid mutations L234A, L235A, and P329G ("P329G LALA"). The "P329G LALA" combination of amino acid substitutions almost completely abolished Fc γ receptor binding of the human IgG1 Fc domain as described in PCT patent application No. WO 2012/130831 a 1. The document also describes methods of making such mutant Fc domains and methods for determining properties thereof, such as Fc receptor binding or effector function. Such antibodies are IgG1 with mutations L234A and L235A or with mutations L234A, L235A and P329G (numbering according to the EU index of Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5 th edition, National Institutes of Health, Bethesda, MD, 1991).

In one aspect, the Fc domain is an IgG4 Fc domain. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising an amino acid substitution at position S228 (Kabat numbering), in particular the amino acid substitution S228P. In a more specific embodiment, the Fc domain is an IgG4 Fc domain comprising the amino acid substitutions L235E and S228P and P329G. This amino acid substitution reduces the in vivo Fab arm exchange of IgG4 antibody (see Stubenrauch et al, Drug Metabolism and Disposition 38, 84-91 (2010)).

Antibodies with extended half-life and improved neonatal Fc receptor (FcRn) binding, responsible for the transfer of maternal IgG to the fetus (Guyer, R.L. et al, J.Immunol.117(1976) 587. quadrature. 593, and Kim, J.K. et al, J.Immunol.24(1994) 2429. quadrature. 2434) are described in US 2005/0014934. Those antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238. 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, for example, a substitution of residue 434 in the Fc region (U.S. patent No. 7,371,826). For further examples of Fc region variants, see also Duncan, a.r. and Winter, g., Nature 322(1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351.

Binding to Fc receptors can be readily determined, for example, by ELISA or by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Suitable such binding assays are described herein. Alternatively, cell lines known to express specific Fc receptors (such as human NK cells expressing Fc γ IIIa receptors) can be used to assess the binding affinity of Fc domains or Fc domain containing cell activating bispecific antigen binding molecules to Fc receptors. The effector function of an Fc domain, or a bispecific antigen binding molecule comprising an Fc domain of the invention, can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. nos. 5,500,362; hellstrom et al, Proc Natl Acad Sci USA 83, 7059-; U.S. Pat. nos. 5,821,337; bruggemann et al, J Exp Med 166, 1351-. Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI for flow cytometry) ^TMNon-radioactive cytotoxicity assay (CellTechnology, inc. mountain View, CA); and Cytotox

Non-radioactive cytotoxicity assay (Promega, Madison, WI)). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in an animal model such as disclosed in Clynes et al, Proc Natl Acad Sci USA 95, 652-.

The following sections describe preferred aspects of bispecific antigen binding molecules of the invention comprising Fc domain modifications that reduce Fc receptor binding and/or effector function. In one aspect, the present invention relates to a bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) (iii) a third Fab fragment, whichCapable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antibody to an Fc receptor, particularly to an fey receptor. In another aspect, the present invention relates to a bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein the Fc domain comprises one or more amino acid substitutions that reduce effector function. In another aspect, the present invention relates to a bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein the Fc domain is compared to a native IgG ₁The Fc domain exhibits reduced binding affinity to Fc receptors, particularly Fc γ receptors, and/or reduced effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC). In another aspect, the present invention relates to a bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor and/or effector function, particularly wherein the one or more amino acid substitutions are at one or more positions selected from the group consisting of: l234, L235 and P329(Kabat EU index numbering). In another aspect, the invention relates to a bispecific antigen binding molecule,which comprises the following steps: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein each subunit in the Fc domain comprises three amino acid substitutions that reduce binding to an activated Fc receptor and/or effector function, wherein the amino acid substitutions are at positions L234A, L235A, and P329G (Kabat EU index numbering). In a particular aspect, the Fc domain belongs to the subclass human IgG1, having the amino acid mutations L234A, L235A, and P329G (numbered according to the Kabat EU index).

Fc domain modification to promote heterodimerization

The bispecific antigen binding molecules of the present invention comprise different antigen binding sites fused to one or the other of the two subunits of the Fc domain, and thus the two subunits of the Fc domain can be comprised in two non-identical polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. In order to increase the yield and purity of the bispecific antigen binding molecules of the invention in recombinant production, it would therefore be advantageous to introduce modifications in the Fc domain of the bispecific antigen binding molecules of the invention that promote the association of the desired polypeptides.

Accordingly, the present invention relates to a bispecific antigen binding molecule comprising: (a) at least one antigen binding domain capable of specifically binding to 4-1BB, (b) at least one antigen binding domain capable of specifically binding to a target cell antigen, and (c) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein the Fc domain comprises a modification that facilitates binding of the first subunit and the second subunit of the Fc domain. The most extensive site of protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one aspect, the modification is in the CH3 domain of the Fc domain.

In a particular aspect, the modification is a so-called "protuberance-into-hole" modification, which includes a "protuberance" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Accordingly, the present invention relates to a bispecific antigen binding molecule wherein in the CH3 domain of the first subunit of the Fc domain, an amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby generating a bulge within the CH3 domain of the first subunit, which bulge is positionable in a cavity within the CH3 domain of the second subunit; whereas in the CH3 domain of the second subunit of the Fc domain, the amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit can be positioned. Accordingly, there is provided a bispecific antigen molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein the first subunit of the Fc domain comprises a protuberance and the second subunit of the Fc domain comprises a pore according to the protuberance-into-pore method. In particular, the amino acid residue having a larger side chain volume is selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W), and the amino acid residue having a smaller side chain volume is selected from the group consisting of: alanine (a), serine (S), threonine (T) and valine (V). In one aspect, in the first subunit of the Fc domain, the threonine residue at position 366 is substituted with a tryptophan residue (T366W); and in the second subunit of the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V); and optionally, additionally in the second subunit of the Fc domain, the threonine residue at position 366 is substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A) (both numbered according to the Kabat EU index). In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (numbering according to the Kabat EU index) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

Protrusion access hole techniques are described, for example, in US 5,731,168; US 7,695,936; ridgway et al, Prot Eng 9, 617. sup. 621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, the method involves introducing a bulge ("protuberance") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the bulge can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. The bulge is constructed by substituting a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities having the same or similar size as the bulge are created in the interface of the second polypeptide by substituting a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

Thus, in one aspect, in the CH3 domain of the first subunit of the Fc domain of the bispecific antigen binding molecule of the invention, an amino acid residue is substituted with an amino acid residue having a larger side chain volume, thereby creating a bulge within the CH3 domain of the first subunit that can be positioned in a cavity within the CH3 domain of the second subunit; whereas in the CH3 domain of the second subunit of the Fc domain an amino acid residue is substituted with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit can be positioned. The projections and cavities can be made by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis. In a particular aspect, in the CH3 domain of the first subunit of the Fc domain, the threonine residue at position 366 is substituted with a tryptophan residue (T366W), while in the CH3 domain of the second subunit of the Fc domain, the tyrosine residue at position 407 is substituted with a valine residue (Y407V). In one aspect, additionally in the second subunit of the Fc domain, the threonine residue at position 366 is substituted with a serine residue (T366S) and the leucine residue at position 368 is substituted with an alanine residue (L368A).

In another aspect, additionally in the first subunit of the Fc domain, the serine residue at position 354 is substituted with a cysteine residue (S354C), and additionally in the second subunit of the Fc domain, the tyrosine residue at position 349 is substituted with a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of disulfide bridges between the two subunits of the Fc domain, thereby further stabilizing the dimer (Carter (2001), J immunological Methods 248, 7-15). In a particular aspect, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

In another aspect, the modifications that facilitate association of the first and second subunits of the Fc domain include modifications that mediate electrostatic steering effects, for example as described in PCT publication WO 2009/089004. Typically, the method involves substituting one or more amino acid residues at the interface of two Fc domain subunits with charged amino acid residues such that homodimer formation becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable.

The C-terminus of the heavy chain of the bispecific antibody as reported herein may be the complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be the shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending in PG. In one of all aspects reported herein, a bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In one embodiment of all aspects reported herein, the bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, numbering according to the EU index of Kabat).

Modification of the CH1/CL Domain

To further improve correct pairing, bispecific antigen binding molecules may comprise amino acid substitutions with different charges (so-called "charged residues"). These modifications were introduced into the crossover or non-crossover CH1 and CL domains. In a particular aspect, the invention relates to a bispecific antigen binding molecule, wherein in at least one of the CL domains the amino acid at position 123 (EU numbering) has been substituted with arginine (R) and the amino acid at position 124 (EU numbering) has been substituted with lysine (K); and wherein in at least one of the CH1 domains, amino acids 147 (EU numbering) and 213 (EU numbering) have been substituted with glutamic acid (E). More specifically, the present invention relates to a bispecific antigen binding molecule wherein in the CL domain of the Fab domain that binds to 4-1BB, the amino acid at position 123 (EU numbering) has been substituted with arginine (R) and the amino acid at position 124 (EU numbering) has been substituted with lysine (K); and wherein in the CH1 domain of the Fab domain that binds 4-1BB, amino acids 147 (EU numbering) and 213 (EU numbering) have been substituted with glutamic acid (E).

Modifications in the Fab domain

The present invention relates to a bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein in a second Fab fragment capable of specific binding to a target cell antigen, (i) the variable regions VL and VH of the Fab light chain and Fab heavy chain are replaced with each other, or (ii) the constant regions CL and CH1 of the Fab light chain and Fab heavy chain are replaced with each other. Thus, bispecific antibodies were prepared according to the crosssmab technique.

WO2009/080252 and Schaefer, W. et al (PNAS,108(2011) 11187-. They significantly reduce the by-products resulting from the mismatch of the light chain against the first antigen and the wrong heavy chain against the second antigen (compared to a method without such domain exchanges).

In one aspect, the invention relates to a bispecific antigen binding molecule, wherein in a second Fab fragment capable of specific binding to a target cell antigen (i) the variable regions VL and VH of the Fab light chain and Fab heavy chain are replaced with each other, or (ii) the constant regions CL and CH1 of the Fab light chain and Fab heavy chain are replaced with each other, wherein in the second Fab fragment the constant domains CL and CH1 are replaced with each other, such that the CH1 domain becomes part of the light chain and the CL domain becomes part of the heavy chain (CH-CL cross mab). In a further aspect, in the second Fab fragment, the variable domains VL and VH are replaced with each other such that the VH domain becomes part of the light chain and the VL domain becomes part of the heavy chain (VH-VL cross). More specifically, in a second Fab fragment capable of specifically binding to a target cell antigen, the variable regions VL and VH of the Fab light chain and Fab heavy chain are replaced with each other such that the VH domain becomes part of the light chain and the VL domain becomes part of the heavy chain.

Polynucleotide

The invention also provides isolated polynucleotides encoding the bispecific antigen binding molecules, or fragments thereof, as described herein.

An isolated polynucleotide encoding a bispecific antigen binding molecule of the invention may be expressed as a single polynucleotide encoding the entire antigen binding molecule, or as multiple (e.g., two or more) polynucleotides that are co-expressed. Polypeptides encoded by the co-expressed polynucleotides may associate via, for example, disulfide bonds or other means to form a functional antigen binding molecule. For example, the light chain portion of an immunoglobulin may be encoded by a separate polynucleotide from the heavy chain portion of an immunoglobulin. When co-expressed, the heavy chain polypeptide will associate with the light chain polypeptide to form an immunoglobulin.

In some aspects, the isolated polynucleotide encodes a polypeptide as described herein comprised in a bispecific molecule according to the invention.

In one aspect, the invention relates to an isolated polynucleotide encoding a bispecific antigen binding molecule comprising: (a) a first Fab fragment capable of specifically binding to 4-1 BB; (b) a second Fab fragment capable of specifically binding to a target cell antigen; (c) a third Fab fragment capable of specifically binding to 4-1 BB; and (d) an Fc domain consisting of a first subunit and a second subunit capable of stable binding, wherein the first Fab fragment and the third Fab fragment capable of specific binding to 4-1BB each comprise: heavy chain variable region (V) _H4-1BB) comprising (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and light chain variable region (V)_L4-1BB) comprising (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the invention is RNA, for example in the form of messenger RNA (mrna). The RNA of the present invention may be single-stranded or double-stranded.

Recombination method

The bispecific antigen binding molecules of the invention can be obtained, for example, by recombinant production. For recombinant production, one or more polynucleotides encoding bispecific antigen binding molecules or polypeptide fragments thereof are provided. One or more polynucleotides encoding the bispecific antigen binding molecule are isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such polynucleotides can be readily isolated and sequenced using conventional methods. In one aspect of the invention, there is provided a vector, preferably an expression vector, comprising one or more of the polynucleotides of the invention. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of the bispecific antigen binding molecule (fragment) and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination/genetic recombination. See, for example, the techniques described in: maniatis et al, Molecula clone, A Laboratory Manual, Cold Spring Harbor LABORATORY, N.Y. (1989); and Ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). The expression vector may be part of a plasmid, virus, or may be a nucleic acid fragment. The expression vector includes an expression cassette into which a polynucleotide encoding the bispecific antigen binding molecule or polypeptide fragment thereof (i.e., the coding region) is cloned in operable association with a promoter and/or other transcriptional or translational control elements. As used herein, a "coding region" is a portion of a nucleic acid that consists of codons that are translated into amino acids. Although the "stop codon" (TAG, TGA or TAA) is not translated into an amino acid, it (if present) can be considered part of the coding region, whereas any flanking sequences, such as promoters, ribosome binding sites, transcription terminators, introns, 5 'and 3' untranslated regions, etc., are not part of the coding region. The two or more coding regions may be present in a single polynucleotide construct (e.g., on a single vector), or in separate polynucleotide constructs (e.g., on separate (different) vectors). In addition, any vector may contain a single coding region, or may contain two or more coding regions, e.g., a vector of the invention may encode one or more polypeptides that are separated into the final protein by proteolytic cleavage post-or post-translationally. In addition, the vectors, polynucleotides or nucleic acids of the invention may encode a heterologous coding region, which is fused or not fused to a polynucleotide encoding a bispecific antigen binding molecule of the invention or a polypeptide fragment thereof, or a variant or derivative thereof. Heterologous coding regions include, but are not limited to, specialized elements or motifs, such as secretion signal peptides or heterologous functional domains. Operable association is when the coding region of a gene product (e.g., a polypeptide) is associated with one or more regulatory sequences in a manner such that expression of the gene product is under the influence or control of the regulatory sequences. Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in transcription of mRNA encoding the desired gene product, and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression control sequences to direct expression of the gene product or with the ability of the gene template to be transcribed. Thus, if a promoter is capable of affecting transcription of the nucleic acid, the promoter region will be operably associated with the nucleic acid encoding the polypeptide. The promoter may be a cell-specific promoter that directs substantial transcription of DNA only in predetermined cells. In addition to promoters, other transcriptional control elements, such as enhancers, operators, repressors, and transcriptional termination signals, may be operably associated with a polynucleotide to direct cell-specific transcription.

Suitable promoters and other transcriptional control regions are disclosed herein. Various transcriptional control regions are known to those skilled in the art. These transcriptional control regions include, but are not limited to, transcriptional control regions that function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegalovirus (e.g., immediate early promoter-binding intron-a), simian virus 40 (e.g., early promoter), and retroviruses (such as, for example, rous sarcoma virus). Other transcriptional control regions include those derived from vertebrationPhysical genes (such as actin, heat shock protein, bovine growth hormone and rabbit)

Globin), and other sequences capable of controlling gene expression in eukaryotic cells. Other suitable transcriptional control regions include tissue-specific promoters and enhancers and inducible promoters (e.g., tetracycline-inducible promoters). Similarly, various translational control elements are known to those of ordinary skill in the art. These translation control elements include, but are not limited to, ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly internal ribosome entry sites, or IRES, also known as CITE sequences). The expression cassette may also include other features, such as an origin of replication, and/or chromosomal integration elements, such as retroviral Long Terminal Repeats (LTRs), or adeno-associated virus (AAV) Inverted Terminal Repeats (ITRs).

The polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions encoding a secretion peptide or signal peptide which direct secretion of the polypeptide encoded by the polynucleotide of the present invention. For example, if secretion of the bispecific antigen binding molecule or polypeptide fragment thereof is desired, a DNA encoding a signal sequence can be placed upstream of a nucleic acid encoding the bispecific antigen binding molecule or polypeptide fragment thereof of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence that is cleaved from the mature protein once the protein chain has been initiated to grow across the rough endoplasmic reticulum export. One of ordinary skill in the art will recognize that polypeptides secreted by vertebrate cells typically have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the translated polypeptide to yield a secreted or "mature" form of the polypeptide. In certain embodiments, a native signal peptide (e.g., an immunoglobulin heavy or light chain signal peptide) is used, or a functional derivative of that sequence that retains the ability to direct secretion of a polypeptide with which it is operably associated. Alternatively, a heterologous mammalian signal peptide or functional derivative thereof may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human Tissue Plasminogen Activator (TPA) or mouse β -glucuronidase.

DNA encoding short protein sequences (e.g., histidine tags) that can be used to facilitate subsequent purification or DNA that helps label the fusion protein can be included within or at the end of the polynucleotide encoding the bispecific antigen binding molecule of the invention or polypeptide fragment thereof.

In another aspect of the invention, host cells comprising one or more polynucleotides of the invention are provided. In certain aspects, host cells comprising one or more vectors of the invention are provided. The polynucleotide and vector may be introgressed, individually or in combination, with any of the features described herein with respect to the polynucleotide and vector, respectively. In one aspect, the host cell comprises (e.g., has been transformed or transfected with) a vector comprising a polynucleotide encoding (part of) the bispecific antigen binding molecule of the invention. As used herein, the term "host cell" refers to any kind of cellular system that can be engineered to produce a fusion protein of the invention or a fragment thereof. Host cells suitable for replicating and supporting the expression of antigen binding molecules are well known in the art. Such cells can be appropriately transfected or transduced with a particular expression vector, and large numbers of vector-containing cells can be grown for seeding large-scale fermentors to obtain sufficient quantities of antigen binding molecules for clinical use. Suitable host cells include prokaryotic microorganisms such as E.coli, or various eukaryotic cells such as Chinese hamster ovary Cells (CHO), insect cells, and the like. For example, the polypeptide may be produced in bacteria, particularly when glycosylation is not required. The polypeptide can be isolated from the bacterial cell paste after expression in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides, including fungi and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of polypeptides having a partially or fully human glycosylation pattern. See Gerngross, Nat Biotech 22, 1409-.

Suitable host cells for the expression (glycosylation) of polypeptides also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified which can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda (Spodoptera frugiperda) cells. Plant cell cultures may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIIES for antibody production in transgenic plants^TMA technique). Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (293 or 293T cells, as described for example in Graham et al, J Gen Virol 36,59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described for example in Mather, Biol Reprod 23, 243-. Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including dhfr-CHO cells (Urlaub et al, Proc Natl Acad Sci USA 77,4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63, and Sp 2/0. For a review of certain mammalian host cell lines suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B.K.C.Lo eds., Humana Press, Totowa, NJ), pp.255-268 (2003). Host cells include cultured cells, such as mammalian cultured cells, yeast cells, insect cells, bacterial cells, and plant cells, to name a few, and also include transgenic animals, transgenic plants, or cultured plants Or cells contained in animal tissues. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell, a Human Embryonic Kidney (HEK) cell, or a lymphocyte (e.g., Y0, NS0, Sp20 cell). Standard techniques for expressing foreign genes in these systems are known in the art. Cells expressing a polypeptide comprising the heavy or light chain of an immunoglobulin can be engineered to also express another immunoglobulin chain, such that the expressed product is an immunoglobulin with a heavy and light chain.

In one aspect, a method of producing a bispecific antigen binding molecule or polypeptide fragment thereof of the invention is provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding the bispecific antigen binding molecule or polypeptide fragment thereof of the invention as provided herein under conditions suitable for expression of the bispecific antigen binding molecule or polypeptide fragment thereof of the invention, and recovering the bispecific antigen binding molecule or polypeptide fragment thereof of the invention from the host cell (or host cell culture medium).

Bispecific molecules of the invention prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those skilled in the art. For affinity chromatography purification, antibodies, ligands, receptors, or antigens that bind to the bispecific antigen binding molecule can be used. For example, for affinity chromatography purification of the fusion protein of the invention, a matrix with protein a or protein G may be used. The antigen binding molecules can be separated using sequential protein a or G affinity chromatography and size exclusion chromatography, essentially as described in the examples. The purity of the bispecific antigen binding molecule or fragment thereof can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like. For example, the expressed bispecific antigen binding molecules described in the examples were shown to be intact and properly assembled as shown by reducing and non-reducing SDS-PAGE.

Measurement of

The physical/chemical properties and/or biological activities of the bispecific antigen binding molecules provided herein can be identified, screened or characterized by various assays known in the art.

1. Affinity assay

The affinity of the bispecific antigen binding molecules, antibodies and antibody fragments provided herein for 4-1BB and target cell antigens can be determined by Surface Plasmon Resonance (SPR) according to the methods set forth in the examples using standard instruments such as BIAcore instruments (GE Healthcare) as well as receptors or target proteins that can be obtained by recombinant expression. The affinity of the bispecific antigen binding molecule for a target cell antigen can also be determined by Surface Plasmon Resonance (SPR) using standard instruments such as BIAcore instruments (GE Healthcare) and receptors or target proteins obtainable by recombinant expression. Specific illustrative and exemplary embodiments for measuring binding affinity are described in example 1.2. According to one aspect, at 25 deg.C

The T100 instrument (GE Healthcare) measures K by surface plasmon resonance_D。

2. Binding assays and other assays

Binding of bispecific antigen binding molecules provided herein to cells expressing the corresponding receptor can be assessed by flow cytometry (FACS) using cell lines expressing the particular receptor or target antigen. In one aspect, the 4-1BB expressing reporter cell line Jurkat-hu4-1BB-NF κ B-luc2 is used in a binding assay. In a further aspect, binding of the antigen-binding molecule to a target cell antigen is demonstrated using a cancer cell line expressing the target cell antigen (e.g., FAP or CEA).

In another aspect, competition assays can be used to identify antigen binding molecules that compete with a particular antibody or antigen binding molecule, respectively, for binding to target or 4-1 BB. In certain embodiments, such competing antigen binding molecules bind to the same epitope (e.g., a linear or conformational epitope) bound by a particular anti-target antibody or a particular anti-4-1 BB antibody. Detailed exemplary methods for locating an epitope to which an antibody binds are provided in: morris (1996), "Epitope Mapping Protocols", from Methods in Molecular Biology Vol.66 (Humana Press, Totowa, NJ).

3. Activity assay

In one aspect, an assay method is provided for identifying bispecific antigen binding molecules that bind to a specific target cell antigen and 4-1BB that is biologically active. Biological activity can include, for example, agonistic signaling through 4-1BB to cells expressing a target cell antigen. Bispecific antigen binding molecules identified by assays having such in vitro biological activities are also provided.

In certain aspects, the bispecific antigen binding molecules of the invention are tested for such biological activity. In addition, methods for detecting cell lysis (e.g., by measuring LDH release), induced apoptosis kinetics (e.g., by measuring caspase 3/7 activity), or apoptosis (e.g., using TUNEL assays) are well known in the art. In addition, the biological activity of such complexes can be assessed by assessing the effect of such complexes on survival, proliferation and lymphokine secretion of various lymphocyte subpopulations such as NK cells, NKT cells or gamma T cells, or their ability to modulate the phenotype and function of antigen presenting cells such as dendritic cells, monocytes/macrophages or B cells.

Pharmaceutical compositions, formulations and routes of administration

In a further aspect, the invention provides pharmaceutical compositions comprising one or more of the bispecific antigen binding molecules provided herein, for example for use in any of the following methods of treatment. In one embodiment, the pharmaceutical composition comprises any one of the bispecific antigen binding molecules provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises any one of the bispecific antigen binding molecules provided herein and at least one additional therapeutic agent as described below.

The pharmaceutical compositions of the invention comprise a therapeutically effective amount of one or more bispecific antigen binding molecules dissolved or dispersed in a pharmaceutically acceptable excipient. The term "pharmaceutically or pharmacologically acceptable" means that the molecular entities and compositions are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other untoward reactions when administered to an animal (e.g., a human) as appropriate. The preparation of Pharmaceutical compositions comprising at least one bispecific antigen binding molecule according to the invention and optionally additional active ingredients will be known to the person skilled in the art in view of this disclosure, as exemplified by Remington's Pharmaceutical Sciences (18 th edition, Mack Printing Company, 1990), which is incorporated herein by reference. Specifically, the composition is a lyophilized formulation or an aqueous solution. As used herein, "pharmaceutically acceptable excipient" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers, and combinations thereof, as known to one of ordinary skill in the art.

Parenteral compositions include those designed for injection (e.g., subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal, or intraperitoneal injection). For injection, the bispecific antigen binding molecules of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks 'solution, ringer's solution or physiological saline. The solution may contain formulating agents (formulations), such as suspending, stabilizing and/or dispersing agents. Alternatively, the bispecific antigen binding molecule may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use. Sterile injectable solutions are prepared by incorporating the antigen-binding molecules of the invention in the required amount in the appropriate solvent with various other ingredients enumerated below, as required. For example, sterility can be readily achieved by filtration through sterile filtration membranes. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and/or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, the preferred methods of preparation are vacuum drying or lyophilization techniques that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium. The liquid medium should be suitably buffered if necessary, and sufficient saline or glucose should first be used to render the liquid diluent isotonic prior to injection. The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept to a minimum at a safe level, for example below 0.5ng/mg protein. Suitable pharmaceutically acceptable excipients include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and/or a non-ionic surfactant, such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, dextran, and the like. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil; or synthetic fatty acid esters such as ethyl oleate or triglycerides; or liposomes.

The active ingredient may be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively); in colloidal drug delivery systems (e.g., liposomes, albumin, microspheres, microemulsions, nanoparticles, and nanocapsules); or in a coarse emulsion. Such techniques are disclosed in Remington's Pharmaceutical Sciences (18 th edition, Mack Printing Company, 1990). Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, which matrices are in the form of shaped articles, e.g., films, or microcapsules. In certain embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate, gelatin or combinations thereof.

Exemplary pharmaceutically acceptable excipients herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (r: (r) ())

Baxter International, Inc.). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases (such as chondroitinase).

Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulations comprising histidine-acetate buffer.

In addition to the compositions previously described, the antigen binding molecules may also be formulated as long acting preparations. Such long acting formulations may be administered by implantation (e.g. subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the fusion protein may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

Pharmaceutical compositions comprising the bispecific antigen binding molecules of the invention may be produced by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes. The pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations which can be used pharmaceutically. Suitable formulations depend on the route of administration chosen.

The bispecific antigen binding molecules can be formulated into compositions in free acid or base, neutral or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or free base. Such pharmaceutically acceptable salts include acid addition salts, for example formed with the free amino groups of the proteinaceous composition, or with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide; or an organic base such as isopropylamine, trimethylamine, histidine or procaine. Pharmaceutically acceptable salts tend to be more soluble in aqueous and other protic solvents than the corresponding free base forms.

The compositions herein may also contain more than one active ingredient necessary for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in an amount effective for the intended purpose.

The formulations to be used for in vivo administration are generally sterile. For example, sterility can be readily achieved by filtration through sterile filtration membranes.

Therapeutic methods and compositions

Any of the bispecific antigen binding molecules provided herein can be used in a method of treatment. For use in a method of treatment, the bispecific antigen binding molecules of the invention may be formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the practitioner.

In one aspect, the bispecific antigen binding molecules of the invention are provided for use as a medicament.

In other aspects, bispecific antigen binding molecules of the invention are provided (i) for stimulating or enhancing T cell responses, (ii) for supporting the survival of activated T cells, (iii) for treating cancer, (iv) for delaying cancer progression, or (v) for prolonging the survival of cancer patients. In a particular aspect, bispecific antigen binding molecules of the invention are provided for the treatment of diseases, in particular for the treatment of cancer.

In certain aspects, bispecific antigen binding molecules of the invention are provided for use in a method of treatment. In one aspect, the invention provides a bispecific antigen binding molecule as described herein for use in treating a disease in an individual in need thereof. In certain aspects, the invention provides bispecific antigen binding molecules for use in a method of treating an individual having a disease, the method comprising administering to the individual a therapeutically effective amount of the bispecific antigen binding molecule. In certain aspects, the disease to be treated is cancer. The subject, patient or "individual" in need of treatment is typically a mammal, more particularly a human.

In one aspect, there is provided a method of (i) stimulating or enhancing a T cell response, (ii) supporting survival of activated T cells, (iii) treating cancer, (iv) delaying cancer progression, or (v) prolonging survival of a cancer patient, wherein the method comprises administering to an individual in need thereof a therapeutically effective amount of a bispecific antigen binding molecule of the invention.

In a further aspect, the invention provides the use of a bispecific antigen binding molecule of the invention for the manufacture or preparation of a medicament for the treatment of a disease in an individual in need thereof. In one aspect, the medicament is for use in a method of treating a disease, the method comprising administering to an individual having the disease a therapeutically effective amount of the medicament. In certain aspects, the disease to be treated is a proliferative disorder, particularly cancer. Examples of cancer include, but are not limited to, bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell cancer, bone cancer, and renal cancer. Other examples of cancer include malignancies, lymphomas (e.g., hodgkin's lymphoma and non-hodgkin's lymphoma), blastoma, sarcomas, and leukemias. Other cell proliferation disorders that can be treated using the bispecific antigen binding molecules or antibodies of the invention include, but are not limited to, tumors located in: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testis, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral nervous system), lymphatic system, pelvis, skin, soft tissue, spleen, chest, and urogenital system. Also included are precancerous conditions or lesions and metastases. In certain embodiments, the cancer is selected from the group consisting of: renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. One skilled in the art will readily recognize that in many cases, bispecific antigen binding molecules or antibodies of the invention may not be curative, but may provide benefits. In some aspects, physiological changes with certain benefits are also considered to have therapeutic benefits. Thus, in some aspects, the amount of a bispecific antigen binding molecule or antibody of the invention that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount".

For the prevention or treatment of disease, the appropriate dosage of the bispecific antigen binding molecules of the invention (when used alone or in combination with one or more additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the particular molecule, the severity and course of the disease, whether the bispecific antigen binding molecule or antibody of the invention is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic intervention, the patient's clinical history and response to the fusion protein, and the discretion of the attending physician. In any case, the practitioner responsible for administration will determine the concentration and appropriate dosage of the active ingredient in the composition for the individual subject. Various dosing schedules are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administrations, and pulsed infusions.

The bispecific antigen binding molecules of the invention are suitable for administration to a patient at one time or in a series of treatments. Depending on the type and severity of the disease, about 1 μ g/kg to 15mg/kg (e.g., 0.1mg/kg-10mg/kg) of the bispecific antigen binding molecule may be an initial candidate dose administered to the patient, e.g., by one or more separate administrations, or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may range from about 1. mu.g/kg to 100mg/kg or more. For repeated administrations over several days or longer, depending on the condition, the treatment will generally continue until the desired suppression of disease symptoms occurs. An exemplary dose of a bispecific antigen binding molecule or antibody of the invention ranges from about 0.005mg/kg to about 10 mg/kg. In other examples, the dose may further include about 1 μ g/kg body weight, about 5 μ g/kg body weight, about 10 μ g/kg body weight, about 50 μ g/kg body weight, about 100 μ g/kg body weight, about 200 μ g/kg body weight, about 350 μ g/kg body weight, about 500 μ g/kg body weight, about 1mg/kg body weight, about 5mg/kg body weight, about 10mg/kg body weight, about 50mg/kg body weight, about 100mg/kg body weight, about 200mg/kg body weight, about 350mg/kg body weight, about 500mg/kg body weight to about 1000mg/kg body weight or more per administration, and any range derivable therein. In examples of ranges derivable from the numbers listed herein, ranges of about 0.1mg/kg body weight to about 20mg/kg body weight, about 5 μ g/kg body weight to about 1mg/kg body weight, and the like, can be administered based on the numbers above. Thus, one or more doses of about 0.5mg/kg, 2.0mg/kg, 5.0mg/kg, or 10mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, such as weekly or every three weeks (e.g., such that the patient receives from about 2 to about 20 or, for example, about 6 doses of the fusion protein). In a particular aspect, the bispecific antigen binding molecule will be administered once every three weeks. An initial higher loading dose may be administered followed by one or more lower doses. However, other dosage regimens may be useful. The progress of the therapy is readily monitored by conventional techniques and assays.

The bispecific antigen binding molecules of the invention will generally be used in an amount effective to achieve the intended purpose. For use in treating or preventing a disorder, the bispecific antigen binding molecules or antibodies of the invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, particularly in light of the detailed disclosure provided herein.

For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, such as cell culture assays. Doses can then be formulated in animal models to achieve IC including as determined in cell culture₅₀Circulating concentration range. Such information can be used to more accurately determine useful doses for humans.

Initial dosages can also be estimated from in vivo data (e.g., animal models) using techniques well known in the art. Administration to humans can be readily optimized by one of ordinary skill in the art based on animal data.

The dose and interval can be adjusted individually to provide plasma levels of the bispecific antigen binding molecule or antibody of the invention sufficient to maintain therapeutic efficacy. The usual patient dose for administration by injection is in the range of about 0.1 to 50 mg/kg/day, usually about 0.1 to 1 mg/kg/day. Therapeutically effective plasma levels can be achieved by administering multiple doses per day. Levels in plasma can be measured, for example, by HPLC.

In the case of topical administration or selective ingestion, the effective local concentration of the bispecific antigen binding molecule or antibody of the invention may not be related to the plasma concentration. One skilled in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

Therapeutically effective doses of the bispecific antigen binding molecules of the invention described herein will generally provide therapeutic benefitsWithout causing significant toxicity. Toxicity and therapeutic efficacy of the fusion proteins can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Cell culture assays and animal studies can be used to determine LD₅₀(dose of 50% of lethal population) and ED₅₀(a therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀. Bispecific antigen binding molecules that exhibit large therapeutic indices are preferred. In one aspect, the bispecific antigen binding molecules or antibodies of the invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages suitable for use in humans. The dose is preferably within a range of circulating concentrations that include ED50 with little or no toxicity. The dosage may vary within this range depending upon a variety of factors, such as the dosage form employed, the route of administration utilized, the condition of the subject, and the like. The exact formulation, route of administration and dosage may be selected by The individual physician according to The condition of The patient (see, e.g., Fingl et al, 1975, in: The pharmaceutical Basis of Therapeutics, Chapter 1, page 1, The entire contents of which are incorporated herein by reference).

The attending physician of a patient treated with a bispecific antibody of the present invention will know how and when to terminate, discontinue or adjust administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is inadequate (excluding toxicity), the attending physician will also know to adjust the treatment to higher levels. The size of the dose administered in the management of the condition of interest will vary with the severity of the condition to be treated, the route of administration, and the like. For example, the severity of a condition can be assessed, in part, by standard prognostic assessment methods. In addition, the dose and possibly the frequency of dosing will also vary according to the age, weight and response of the individual patient.

Other Agents and treatments

The bispecific antigen binding molecules of the invention may be administered alone or in combination with one or more other agents in therapy. For example, a bispecific antigen binding molecule or antibody of the invention can be administered in combination with at least one additional therapeutic agent. The term "therapeutic agent" includes any agent that can be administered for the treatment of a symptom or disease in an individual in need of such treatment. Such additional therapeutic agents may comprise any active ingredient suitable for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anti-cancer or chemotherapeutic agent, such as a microtubule disrupting agent, an anti-metabolite, a topoisomerase inhibitor, a DNA intercalating agent, an alkylating agent, an anthracycline, a hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor apoptosis, or an anti-angiogenic agent. In certain aspects, the additional therapeutic agent is an immunomodulator, cytostatic, cell adhesion inhibitor, cytotoxic or cytostatic agent, apoptosis activator, or an agent that increases the sensitivity of a cell to an apoptosis-inducing agent.

In one aspect, the bispecific antigen binding molecules of the invention are administered in combination with chemotherapeutic agents, radiation therapy, and/or other agents for cancer immunotherapy. The chemotherapeutic agent is an anti-cancer agent as defined above. Alternatively, the chemotherapeutic agent is selected from the group consisting of: nucleotide analogs (e.g., azacitidine, capecitabine, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, or methotrexate), platinum-based drugs (e.g., carboplatin, cisplatin, or oxaliplatin), taxanes (e.g., paclitaxel, docetaxel, Abraxane, or taxotere), alkylating agents (e.g., cyclophosphamide, chlorambucil, dacarbazine, or temozolomide), anthracyclines (e.g., doxorubicin or idarubicin), topoisomerase I inhibitors (e.g., irinotecan or topotecan), topoisomerase II (e.g., etoposide or teniposide), kinase inhibitors (e.g., erlotinib, imatinib, vemurafenib, or vegie), retinoids, histone deacetylase inhibitors, and vinca alkaloids. Other agents for cancer immunotherapy include, for example, agents that block the PD-L1/PD-1 interaction, such as a PD1 antibody (e.g., pembrolizumab or nivolumab) or a PD-L1 antibody (e.g., atlizumab). Bispecific antigen binding molecules of the invention may also be used in combination with radiotherapy.

Such other agents are suitably present in combination in an amount effective for the intended purpose. The effective amount of such other agents will depend on the amount of fusion protein used, the type of disorder or treatment, and other factors discussed above. The bispecific antigen binding molecules or antibodies of the invention are typically used in the same dosages and routes of administration as described herein, or about 1% to 99% of the dosages described herein, or in any dosage and by an empirically/clinically determined appropriate route.

Such combination therapies described above encompass combined administration (where two or more therapeutic agents are contained in the same composition or separate compositions), as well as separate administration, in which case administration of the bispecific antigen binding molecule or antibody of the invention can occur prior to, concurrently with, and/or after administration of additional therapeutic agents and/or adjuvants. In one aspect, administration of the bispecific antigen binding molecule and administration of the additional therapeutic agent occur within about 1 month or within about 1, 2, or 3 weeks or within about 1, 2, 3, 4, 5, or 6 days of each other.

Article of manufacture

In another aspect of the invention, an article of manufacture is provided that contains materials useful for the treatment, prevention and/or diagnosis of the above-mentioned conditions. The article of manufacture comprises a container and a label or package insert (package insert) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, Intravenous (IV) solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains the composition, alone or in combination with another composition effective for treating, preventing and/or diagnosing the condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a bispecific antigen binding molecule of the invention.

The label or package insert indicates that the composition is for use in treating the selected condition. In addition, an article of manufacture can comprise (a) a first container comprising a composition comprising a bispecific antigen binding molecule of the present invention; and (b) a second container containing a composition comprising an additional cytotoxic agent or other therapeutic agent. The article of manufacture of this embodiment of the invention may further comprise a package insert indicating that the composition is useful for treating a particular condition.

Alternatively or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, ringer's solution, and dextrose solution. The article of manufacture may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Table B (sequence):

for all nucleotide sequences, no corresponding stop codon sequence is present.

Aspects of the invention

The following numbered paragraphs describe various aspects of the invention:

1. a bispecific antigen binding molecule comprising:

(a) a first Fab fragment capable of specifically binding to 4-1 BB;

(c) a third Fab fragment capable of specifically binding to 4-1 BB; and

(d) an Fc domain consisting of a first and a second subunit capable of stable binding, wherein a second Fab fragment (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the first Fab fragment (a), which in turn is fused at its C-terminus to the N-terminus of the first Fc domain subunit, and a third Fab fragment (C) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit, and wherein in the second Fab fragment capable of specific binding to a target cell antigen, (i) the variable domains VL and VH are replaced with each other, or (ii) the constant domains CL and CH1 are replaced with each other.

2. The bispecific antigen binding molecule of paragraph 1, wherein the bispecific antigen binding molecule provides bivalent binding to 4-1BB and monovalent binding to a target cell antigen.

3. The bispecific antigen binding molecule according to

paragraph

1 or 2, wherein the Fc domain consisting of the first and second subunits capable of stable binding is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain.

4. The bispecific antigen binding molecule according to any one of paragraphs 1 to 3, wherein the first subunit of the Fc domain comprises a protuberance and the second subunit of the Fc domain comprises a pore according to the protuberance-into-pore method.

5. The bispecific antigen binding molecule according to any one of paragraphs 1 to 4, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, in particular the amino acid mutations L234A, L235A and P329G (numbering according to the EU index of Kabat).

6. The bispecific antigen binding molecule of any one of paragraphs 1 to 5, wherein the first and third Fab fragments capable of specific binding to 4-1BB are identical.

7. The bispecific antigen-binding molecule of any one of paragraphs 1 to 6, wherein is capable of specifically binding to 4-1BBThe first and third Fab fragments each comprise: heavy chain variable region (V)_H4-1BB) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and light chain variable region (V)_L4-1BB) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

8. The bispecific antigen binding molecule of any one of paragraphs 1 to 7, wherein the first and third Fab fragments capable of specific binding to 4-1BB each comprise: heavy chain variable region (V)_H4-1BB) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 7; and light chain variable region (V)_L4-1BB) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 8.

9. The bispecific antigen binding molecule according to any one of paragraphs 1 to 8, wherein in the constant domains CL of the first and third Fab fragments capable of specifically binding to 4-1BB, the amino acid at position 124 is substituted with lysine (K) (numbered according to the Kabat EU index) and the amino acid at position 123 is substituted with arginine (R) or lysine (K) (numbered according to the Kabat EU index), and wherein in the constant domains CH1 of the first and third Fab fragments capable of specifically binding to 4-1BB, the amino acid at position 147 is substituted with glutamic acid (E) (numbered according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbered according to the Kabat EU index).

10. The bispecific antigen binding molecule according to any one of paragraphs 1 to 9, wherein in the second Fab fragment capable of specific binding to a target cell antigen the variable domains VL and VH of the Fab light chain and Fab heavy chain are substituted for each other.

11. The bispecific antigen binding molecule according to any one of paragraphs 1 to 10, wherein the second Fab fragment is capable of specifically binding to a target cell antigen selected from the group consisting of: fibroblast Activation Protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, and CD 33.

12. The bispecific antigen binding molecule of any one of paragraphs 1 to 11, wherein the second Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to Fibroblast Activation Protein (FAP).

13. The bispecific antigen binding molecule according to any one of paragraphs 1 to 12, wherein the Fab fragment capable of specific binding to Fibroblast Activation Protein (FAP) comprises:

(a) heavy chain variable region (V)_HFAP) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:9, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:10, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 11; and light chain variable region (V) _LFAP) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:12, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:13, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 14; or

14. The bispecific antigen binding molecule according to any one of paragraphs 1 to 13, wherein the Fab fragment capable of specific binding to Fibroblast Activation Protein (FAP) comprises:

(a) heavy chain variable region (V)_HFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 21; and light chain variable region (V)_LFAP) comprising the same as S22, an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence; or

15. The bispecific antigen binding molecule according to any one of paragraphs 1 to 11, wherein the Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA).

16. The bispecific antigen binding molecule according to any one of paragraphs 1 to 11 or 15, wherein the Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises:

(c) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:41, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:42(ii) a sequence, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 43; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:44, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:45, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 46; or

(d) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:49, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:50, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 51; and light chain variable region (V) _LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:52, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:53, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54.

17. The bispecific antigen binding molecule according to any one of paragraphs 1 to 11 or 15 or 16, wherein the Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises:

(c) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 47; and light chain variable region (V) _LCEA) comprising at least about 95%, 96%, 97%, 98%, 99% or 100% phase with the amino acid sequence of SEQ ID NO 48The same amino acid sequence; or

(d) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 56.

18. The bispecific antigen binding molecule of any one of paragraphs 1 to 11, wherein the Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to CD 19.

19. The bispecific antigen binding molecule of any one of paragraphs 1 to 11 or 18, wherein the Fab fragment capable of specifically binding to CD19 comprises:

20. The bispecific antigen binding molecule of any one of paragraphs 1 to 11 or 18 or 19, wherein the Fab fragment capable of specifically binding to CD19 comprises: heavy chain variable region (V)_HCD19) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 63; and light chain variable region (V)_LCD19) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 64.

21. A polynucleotide encoding the bispecific antigen binding molecule according to any one of paragraphs 1 to 20.

22. A host cell comprising the polynucleotide according to paragraph 21.

23. A method of producing the bispecific antigen binding molecule according to any one of paragraphs 1 to 20, comprising culturing a host cell according to paragraph 22 under conditions suitable for expression of the bispecific antigen binding molecule.

24. A pharmaceutical composition comprising the bispecific antigen binding molecule according to any one of paragraphs 1 to 20 and at least one pharmaceutically acceptable excipient.

25. The pharmaceutical composition according to paragraph 24, for use in the treatment of cancer.

26. The bispecific antigen binding molecule according to any one of paragraphs 1 to 20 or the pharmaceutical composition according to paragraph 24 for use as a medicament.

27. The bispecific antigen binding molecule according to any one of paragraphs 1 to 20, for use

(i) The stimulation of the T-cell response is achieved,

(ii) support the survival of the activated T cells,

(iii) the medicine can be used for treating cancer,

(iv) delay cancer progression, or

(v) Prolonging the survival time of cancer patients.

28. The bispecific antigen binding molecule according to any one of paragraphs 1 to 20 or the pharmaceutical composition according to paragraph 24 for use in the treatment of cancer.

29. The bispecific antigen binding molecule for use in treating cancer according to any one of paragraphs 1 to 20, wherein the bispecific antigen binding molecule is administered in combination with a chemotherapeutic agent, radiation therapy and/or other agent for cancer immunotherapy.

30. Use of the bispecific antigen binding molecule according to any one of paragraphs 1 to 20 or the pharmaceutical composition according to paragraph 24 in the manufacture of a medicament for the treatment of cancer or an infectious disease.

31. A method of inhibiting growth of a tumor cell in an individual, comprising administering to the individual an effective amount of the bispecific antigen binding molecule according to any one of paragraphs 1 to 20 or the pharmaceutical composition according to paragraph 24, to inhibit growth of the tumor cell.

32. A method of treating cancer or an infectious disease, comprising administering to an individual a therapeutically effective amount of the bispecific antigen binding molecule according to any one of paragraphs 1 to 20 or the pharmaceutical composition according to paragraph 24.

***

Examples of the invention

The following are examples of the methods and compositions of the present invention. It is to be understood that various other embodiments may be practiced given the general description provided above.

Recombinant DNA technology

DNA is manipulated using standard methods, such as those described in Sambrook et al, Molecular cloning: A laboratory Manual; cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions. General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in the following references: kabat, E.A. et al, (1991) Sequences of Proteins of Immunological Interest, fifth edition, NIH Publication No 91-3242.

DNA sequencing

The DNA sequence was determined by double-strand sequencing.

Gene synthesis

The desired gene segments were generated by PCR using appropriate templates, or were synthesized from synthetic oligonucleotides and PCR products by automated gene synthesis from Geneart AG (Regensburg, Germany). In the case where the exact gene sequence is not available, oligonucleotide primers are designed based on the sequence of the closest homolog and the gene is isolated by RT-PCR from RNA derived from the appropriate tissue. Gene segments flanked by single restriction enzyme cleavage sites were cloned into standard cloning/sequencing vectors. Plasmid DNA was purified from the transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene segments with appropriate restriction sites were designed to allow subcloning into the corresponding expression vectors. All constructs were designed with a 5' DNA sequence encoding a leader peptide that targets a protein secreted by eukaryotic cells.

Protein purification

The protein was purified from the filtered cell culture supernatant according to standard protocols. Briefly, antibodies were applied to a protein a sepharose column (GE healthcare) and washed with PBS. Elution of the antibody was achieved at pH 2.8, immediately followed by neutralization of the sample. Aggregated proteins were separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20mM histidine, 150mM NaCl (pH 6.0). The monomeric antibody fractions are combined, concentrated (if necessary) using, for example, a MILLIPORE Amicon Ultra (30MWCO) centrifugal concentrator, frozen and stored at-20 ℃ or-80 ℃. Portions of the sample are provided for subsequent protein analysis and analytical characterization, for example by SDS-PAGE, Size Exclusion Chromatography (SEC), or mass spectrometry.

SDS-PAGE

Use according to manufacturer's instructions

Pre-gel systems (Invitrogen). Specifically, 10% or 4-12% is used

Bis-TRIS precast gel (pH 6.4) and

MES (reducing gel, having

Antioxidant electrophoresis buffer additive) or MOPS (non-reducing gel) electrophoresis buffer.

Analytical size exclusion chromatography

Size Exclusion Chromatography (SEC) for determination of aggregation and oligomeric state of the antibody was performed by HPLC chromatography. Briefly, protein A purified antibody was applied to 300mM NaCl, 50mM KH on an Agilent HPLC 1100 system ₂PO₄/K₂HPO₄Tosoh TSKgel G3000SW column in pH 7.5, or applied to a Dionex HPLC systemSuperdex 200 column in 2 XPBS (GE Healthcare). Eluted protein was quantified by UV absorbance and peak area integration. BioRad gel filtration standards 151-1901 were used as standards.

Mass spectrometry

This section describes the characterization of multispecific antibodies with either VH/VL or CH/CL exchange (CrossMab), with emphasis on correct assembly of the multispecific antibodies. The expected primary structure was analyzed by electrospray ionization mass spectrometry (ESI-MS) of deglycosylated intact CrossMab and deglycosylated/plasmin digested or alternatively deglycosylated/restricted LysC digested CrossMab.

Crossmab was deglycosylated with N-glycosidase F in phosphate or Tris buffer at 37 ℃ at a protein concentration of 1mg/ml for up to 17 h. Plasmin or limited lysc (roche) digestion was performed with 100 μ g deglycosylated VH/VL CrossMab in Tris buffer pH 8 at 120 hours and at 37 ℃ for 40min, respectively. Prior to mass spectrometry, the samples were desalted via HPLC on a Sephadex G25 column (GE Healthcare). The total mass was determined via ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

Example 1

Preparation, purification and characterization of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to FAP

1.1 formation of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to FAP

A method for making a bispecific agonistic 4-1BB antibody with bivalent binding to 4-1BB and monovalent binding to FAP is shown in FIG. 1. This construct is also known as the head-to-head (H2H)2+1 form or 2+ 1H 2H 4-1BB (20H4.9)/FAP (4B9) P329GLALA IgG1 or 4-1BB (20H.9) XFAP 2+ 1H 2H.

The first heavy chain HC1 of the construct consisted of: VHCH1 of anti-4-1 BB binding agent (clone 20H4.9) followed by Fc wells. The second heavy chain HC2 consisted of VHCH1 followed by anti-4-1 BB (clone 20H4.9) and VLCH1 with Fc-promulgated anti-FAP binder (clone 4B9 in cross Fab form). The formation and preparation of FAP binder 4B9 is described in WO 2012/020006 a2, which is incorporated herein by reference. For the 4-1BB binder, the VH and VL sequences of clone 20H4.9 were obtained according to US 7,288,638B 2 or US 7,659,384B 2. The two heavy chains combine to form a heterodimer, which includes one FAP-binding cross Fab and two 4-1 BB-binding fabs (fig. 1).

To improve correct pairing, the following mutations have been introduced in the CH-CL of the anti-4-1 BB Fab molecule: E123R and Q124K in CL and K147E and K213E in CH 1. The second light chain LC2 of the anti-FAP binding agent (clone 4B9) consists of VHCL (crossed Fab).

Using a bulge-entry-hole technique, heterodimers were formed by introducing Y349C/T366S/L368A/Y407V mutations in the first heavy chain HC1 (Fc-hole heavy chain) and by introducing S354C/T366W mutations in the second heavy chain HC2 (Fc-bulge heavy chain).

Pro329Gly, Leu234Ala and Leu235Ala mutations were introduced into the constant regions of the protuberance and pore heavy chains to eliminate binding to Fc γ receptors according to the method described in international patent application publication No. WO2012/130831a 1.

The amino acid sequences of bispecific antibody 4-1BB (20H4.9)/FAP (4B9) P329GLALA IgG 12 +1(H2H) can be found in Table 1.

Table 1: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-FAP human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H 4-1BB (20H4.9)/FAP (4B9) P329GLALA IgG1)

1.2 production of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to FAP IgG-like proteins were produced in HEK293 EBNA or CHO EBNA cells

Antibodies and bispecific antibodies were formed by transient transfection of HEK293 EBNA cells or CHO EBNA cells. The cells were centrifuged and then the pre-warmed CD CHO medium (Thermo Fisher, Cat. No. 10743029) was used in place of the original medium. General watchDacarbazones were mixed in CD CHO medium, PEI (polyethyleneimine, Polysciences, Inc, cat. No. 23966-1) was added, the solution was vortex mixed, and incubated at room temperature for 10 minutes. Then, the cells (2Mio/ml) were mixed with the carrier/PEI solution, transferred to flasks, and placed in a shaking incubator at 5% CO ₂Was incubated at 37 ℃ for 3 hours under the atmosphere of (2). After incubation, Excell medium (W.Zhou and A.Kantardjieff, Mammalian Cell Cultures for Biologics management, DOI: 10.1007/978-3-642-54050-9; 2014) containing supplements (80% of the total volume) was added. 1 day after transfection, supplement (feed, 12% of total volume) was added. After 7 days, the cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter), and the protein was purified from the harvested supernatant using standard methods as shown below.

Production of IgG-like proteins in CHO K1 cells

Alternatively, the antibodies and bispecific antibodies described herein were prepared by Evitria using its proprietary vector system by conventional (non-PCR-based) cloning techniques and using suspension-adapted CHO K1 cells (originally received from ATCC and adapted for serum-free growth in suspension culture of Evitria). During the production process, Evtria used its proprietary animal component-free and serum-free medium (eviGrow and eviMake2) and its proprietary transfection reagent (eviFect). The cell supernatant was harvested by centrifugation and subsequent filtration (0.2 μm filter) and the protein was purified from the harvested supernatant using standard methods.

Purification of IgG-like proteins

The protein was purified from the filtered cell culture supernatant according to standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants using Protein A affinity chromatography (equilibration buffer: 20mM sodium citrate, 20mM sodium phosphate, pH 7.5; elution buffer: 20mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0, followed by immediate neutralization of the pH of the sample. By centrifugation (Millipore)

ULTRA-15, Cat No.: UFC903096) and then separating the aggregated from the monomeric protein using size exclusion chromatography in 20mM histidine, 140mM sodium chloride (pH 6.0).

Analysis of IgG-like proteins

The concentration of the purified Protein was determined by measuring the absorbance at 280nm, using the mass extinction coefficient calculated on the basis of the amino acid sequence, according to the method described by Pace et al (Protein Science,1995,4, 2411-1423). The purity and molecular weight of the proteins were analyzed by CE-SDS in the presence and absence of reducing agents using LabChipGXII (Perkin Elmer). The aggregate content was determined by HPLC chromatography at 25 ℃ using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) in running buffer (25 mM K, respectively) ₂HPO₄125mM NaCl, 200mM L-arginine hydrochloride (pH 6.7) or 200mM KH₂PO₄250mM KCl (pH 6.2)).

TABLE 2 Biochemical analysis of anti-4-1 BB, anti-FAP huIgG 1P 329GLALA antigen-binding molecules with 2-2+ 1H 2H

Preparation of antigen and screening tools human 4-1BB Fc (kih):

the DNA sequence encoding the extracellular domain of human 4-1BB (synthesized as per Q07011) was subcloned into a framework comprising the human IgG1 heavy chain CH2 and CH3 domains on the knob. An AcTEV protease cleavage site was introduced between the extracellular domain of the antigen and the Fc of human IgG 1. An Avi tag for directed biotinylation was introduced at the C-terminus of the antigen Fc protrusion. An antigen Fc projection chain comprising the S354C/T366W mutation binds to an Fc pore chain comprising the Y349C/T366S/L368A/Y407V mutation to form a heterodimer comprising a single copy of a 4-1BB ectodomain chain, thereby forming a monomeric form of an Fc linked antigen. Table 3 shows the amino acid sequence of the antigen Fc fusion construct.

Table 3: amino acid sequence of a monomeric antigen Fc (kih) fusion molecule (produced by combining one Fc pore chain with one antigen Fc protuberance chain)

1.3 preparation of bispecific antibody with bivalent binding to 4-1BB and monovalent binding to FAP, wherein VH and VL of FAP are fused to the C-terminus of the heavy chain, used as control

Bispecific agonistic 4-1BB antibodies with bivalent binding to 4-1BB and monovalent binding to FAP, also known as 4-1BB (20H4.9)/FAP (4B9) P329GLALA IgG 12 +1 VH/VL (C-terminus), were prepared, with the VH and VL of the FAP (4B9) binding agent fused to the C-terminus of each heavy chain, respectively. Using a bulge-entry-hole technique, heterodimers were formed by introducing the S354C/T366W mutation in the first heavy chain HC1(Fc bulge heavy chain) and the Y349C/T366S/L368A/Y407V mutation in the second heavy chain HC2(Fc hole heavy chain).

In this example, the first heavy chain HC1 of the construct consisted of: VHCH1 of an anti-4-1 BB binding agent (clone 20H4.9) followed by an Fc bulge was fused at its C-terminus to the VL of an anti-FAP binding agent. The second heavy chain HC2 consisted of an anti-4-1 BB VHCH1 followed by an Fc well, fused at its C-terminus to the VH of an anti-FAP binder (clone 4B 9). The two heavy chains combine to form a heterodimer comprising one FAP-binding moiety and two 4-1 BB-binding fabs.

The amino acid sequences of 2+1 anti-4-1 BB (anti-FAP construct comprising a-FAP VH fused to an Fc-protuberance heavy chain and VL fused to an Fc-pore heavy chain) can be found in table 4.

Table 4: bispecific bivalent anti-4-1 BB/monovalent anti-FAP human IgG 1P 329GLALA antigen binding molecule (construct, comprising a-FAP VH fused to Fc pore chain and VL fused to Fc protrusion chain, referred to as 2+1 VH/VL)

Table 5: biochemical analysis of bispecific antigen-binding molecules with bivalent binding to 4-1BB and monovalent binding to FAP (2+1 VH/VL 4-1BB/FAP human IgG 1P 329 GLAL)

1.4 preparation of bispecific antibodies with bivalent binding to 4-1BB and non-targeting VH and VL moieties (DP47 germline control), in which VH and VL of DP47 are fused to the C-terminus of the heavy chain (control)

Bispecific agonistic 4-1BB antibodies with bivalent binding to 4-1BB and a monovalent non-targeted DP47 germline control, also known as 4-1BB (20H 4.9)/non-targeted (DP47) P329GLALA IgG 12 +1 VH/VL (C-terminus), were prepared, where VH and VL of the non-binding clone (DP47) were fused at the C-terminus of each heavy chain, respectively. Using a bulge-entry-hole technique, heterodimers were formed by introducing the S354C/T366W mutation in the first heavy chain HC1(Fc bulge heavy chain) and the Y349C/T366S/L368A/Y407V mutation in the second heavy chain HC2(Fc hole heavy chain).

In this example, the first heavy chain HC1 of the construct consisted of: VHCH1 of anti-4-1 BB binding agent (clone 20H4.9) followed by an Fc bulge was fused at its C-terminus to VL of DP 47. The second heavy chain HC2 consists of anti-4-1 BB VHCH1 followed by an Fc pore, fused at its C-terminus to the VH of DP 47. The two heavy chains combine to form a heterodimer, which includes DP47 (instead of FAP binding moiety and two 4-1BB binding fabs).

The amino acid sequences of 2+1 anti-4-1 BB (non-targeting (DP47) constructs comprising a-DP47VH fused to the Fc knob heavy chain and VL fused to the Fc pore heavy chain) can be found in table 6.

Table 6: bispecific bivalent anti-4-1 BB/non-targeting (DP47) human IgG 1P 329GLALA antigen binding molecules (constructs comprising DP47VH fused to Fc pore chain and DP47 VL fused to Fc bulge chain, referred to as C-terminal)

Example 2

Preparation, purification and characterization of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to CEA

2.1 formation and production of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to CEA

Bispecific agonistic 4-1BB antibodies with bivalent binding to 4-1BB and monovalent binding to CEA can also be prepared by substituting anti-FAP cross Fab for anti-CEA cross Fab. This construct is also known as the head-to-head (H2H)2+1 form.

The first heavy chain HC1 of this construct consisted of: VHCH1 of anti-4-1 BB binding agent (clone 20H4.9) followed by Fc wells. The second heavy chain HC2 consisted of VHCH1 followed by anti-4-1 BB (clone 20H4.9) and VLCH1 with Fc raised anti-CEA binding agent (in cross Fab form). Parent CEA binding agent A5B7 is described in WO 92/01059. The sequence of CEA binding agent MFE23 is described in WO 2007/071422. For the 4-1BB binder, the VH and VL sequences of clone 20H4.9 were obtained according to US 7,288,638B 2 or US 7,659,384B 2. The two heavy chains bind to form a heterodimer, which includes one CEA-binding cross Fab and two 4-1 BB-binding Fab (FIG. 1).

To improve correct pairing, the following mutations have been introduced in the CH-CL of the anti-4-1 BB Fab molecule: E123R and Q124K in CL and K147E and K213E in CH 1. The second light chain LC2 of the anti-CEA binding agent consists of VHCL (cross Fab).

Bispecific 2+ 1H 2H anti-4-1 BB anti-CEA huIgG 1P 329GLAL antibody was produced as described in example 1.2 for the 2+1 anti-4-1 BB anti-FAP huIgG 1P 329GLAL antibody.

The amino acid sequences of the bispecific 4-1BB (20H4.9)/CEA (A5B7) P329GLALA IgG 12 +1(H2H) antibodies can be found in Table 7, while the amino acid sequences of the bispecific 4-1BB (20H4.9)/CEA (MFE23) P329GLALA IgG 12 +1(H2H) antibodies can be found in Table 8.

Proteins were produced and purified as described in example 1.2.

Table 7: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (A5B7) human IgG 1P 329GLALA antibody (2+ 1H 2H)

Table 8: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (MFE23) human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H)

Other bispecific agonistic 4-1BB antibodies with bivalent binding to 4-1BB and monovalent binding to CEA can be prepared by cloning anti-CEA (T84.66-LCHA) or cloning anti-CEA (CH1A1A 98/99 SF1) with an anti-CEA binding agent. The amino acid sequences of the bispecific 4-1BB (20H4.9) x CEA (T84.66-LCHA) P329 GLAA IgG 12 +1(H2H) antibodies can be found in Table 9, while the amino acid sequences of the bispecific 4-1BB (20H4.9) x CEA (CH1A1A 98/99 SF1) P329 GLAA IgG 12 +1(H2H) antibodies can be found in Table 10.

Table 9: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (T84.66-LCHA) human IgG 1P 329 GLAA antibody (2+ 1H 2H)

Table 10: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (CH1A1A 98/992F 1) human IgG 1P 329 GLAA antigen-binding molecule (2+ 1H 2H)

2.2 formation of humanized variants of anti-CEA antibody A5B7

2.2.1 methods

anti-CEA antibody A5B7 is disclosed by, for example, M.J.Banfield et al (Proteins 1997,29(2),161-171), and its structure can be found in PDB ID:1CLO in Protein Structure database (PDB) (www.rcsb.org, H.M.Berman et al, The Protein Data Bank, Nucleic Acids Research, 2000, 28, 235-242). The entries include heavy and light chain variable domain sequences. To identify a suitable human acceptor framework in the humanization process of the anti-CEA binding agent A5B7, a classical approach was taken, namely to find an acceptor framework with high sequence homology, graft the CDRs onto this framework, and evaluate the conceivable back-mutations. More specifically, the effect of each amino acid difference of the identified framework and the parent antibody on the structural integrity of the binding agent is judged, and back mutations towards the parent sequence are introduced as appropriate. Structural assessment Fv region homology models based on the parental antibody and its humanized versions were created by an internal antibody structural homology modeling tool, which was implemented using the Biovia Discovery Studio Environment version 4.5.

2.2.2 selection of acceptor frameworks and Adaptation thereof

The acceptor framework was selected as shown in table 11 below:

table 11: acceptor framework

The CDR3 post-framework region was adapted from the human J element germline IGJH6 for the heavy chain and the sequence was similar to the kappa J element IGKJ2 for the light chain.

Based on structural considerations, back mutations were introduced at positions 93 and 94 of the heavy chain from the human acceptor framework to the amino acids in the parent binding agent.

2.2.3 VH and VL regions of the resulting humanized CEA antibody

The VH domain of the resulting humanized CEA antibody can be seen in table 12 below, and the VL domain of the resulting humanized CEA antibody can be seen in table 13 below.

Table 12: amino acid sequence of VH Domain of humanized CEA antibody (based on human acceptor framework IGHV3-23 or IGHV3-15)

For the heavy chain, the initial variant 3-23A5-1 was found to be suitable for use in a binding assay (but exhibited slightly less binding capacity than the parent murine antibody) and was selected as the starting point for further modification. Variants based on IGHV3-15 showed lower binding activity compared to the humanized variant 3-23A 5-1.

To restore the full binding activity of the parent chimeric antibody, variants 3-23A5-1A, 3-23A5-1C, and 3-23A5-1D were created. The length of the variant 3-23A5-1, CDR-H2, was also tested for adaptation to the human receptor sequence, but the construct lost binding activity altogether. Due to the putative deamidation hot spot in CDR-H2(Asn53-Gly54), we changed the motif to Asn53-Ala 54. Another possible hotspot Asn73-Ser74 was back mutated to Lys73-Ser 74. Thus, variant 3-23A5-1E was created.

Table 13: amino acid sequence of VL domain of humanized CEA antibody (based on human receptor framework IGKV 3-11).

The light chain is humanized based on the human IGKV3-11 acceptor framework. In the series a5-L1 to a5-L4, it was understood that the variant a5-L1 showed good binding activity (but slightly lower than the parent antibody). Partial humanization of CDR-L1 (variant A5-L2; Kabat positions 30 and 31) completely abolished binding. Likewise, humanization of CDR-H2 (variant A5-L3; Kabat positions 50 to 56) also completely abolished binding. Position 90 (variant A5-L4) has a significant contribution to the binding properties. Histidine at this position is important for binding. Thus, variant A5-L1 was selected for further modification.

The series a5-L1A through a5-L1D solved the problem of back mutations required to restore the full binding potential of the parental chimeric antibody. Variant a5-L1A showed that the Kabat positions 1, 2, whole frame 2 and the back-mutations at Kabat position 71 did not increase any more binding activity. Variants A5-L1B and A5-L1C mapped a subset of those positions and confirmed that they did not alter binding properties. The variant A5-L1D, which had back mutations at Kabat positions 46 and 47, showed the best binding activity.

2.2.4 selection of humanized A5B7 antibodies

Based on novel humanized variants of VH and VL, the novel CEA antibody was expressed as a huIgG1 antibody with effector-silenced Fc (P329G; L234, L235A) to eliminate binding to Fc γ receptor according to the method described in WO 2012/130831 a 1; they were tested for binding to CEA expressed on MKN45 cells and compared to the corresponding parent murine A5B7 antibody.

Table 14: VH/VL combinations expressed as huIgG1_ LALA _ PG antibodies

	A5-L1A	A5-L1B	A5-L1C	A5-L1D
					3-23A5-1A	P1AE2164	P1AE2165	P1AE2166	P1AE2167
3-23A5-1C	-	-	P1AE2176	P1AE2177
					3-23A5-1D	P1AE2179	-	P1AE2181	P1AE2182

MKN45(DSMZ ACC 409) is a human gastric adenocarcinoma cell line expressing CEA. Cells were cultured in high grade RPMI + 2% FCS + 1% Glutamax. The viability of MKN-45 cells was examined and the cells were resuspended and adjusted to a density of 1Mio cells/ml. 100 μ l of this cell suspension (containing 0.1Mio cells) was seeded into a 96-well round bottom plate. The well plates were centrifuged at 400Xg for 4min and the supernatant was removed. Then 40. mu.l of diluted antibody or FACS buffer was added to the cells and incubated at 4 ℃ for 30 minutes. After incubation, cells were washed twice with FACS buffer (150 μ l per well). Then 20. mu.l of diluted secondary PE anti-human Fc specific secondary antibody (109-116-170, Jackson ImmunoResearch) was added to the cells. The cells were incubated at 4 ℃ for a further 30 minutes. To remove unbound antibody, cells were washed twice more with FACS buffer (150 μ Ι per well). To immobilize the cells, 100. mu.l of FACS buffer (containing 1% PFA) was added to the wells. Before measurement, cells were resuspended in 150. mu.l FACS buffer. Fluorescence was measured using a BD flow cytometer.

Figure 7 shows binding curves for humanized A5B7 variants. All tested binding agents were able to bind MKN45 cells, but the binding capacity was slightly reduced compared to the parent A5B7 antibody. Among all variants tested, clone P1AE2167 has the best binding capacity and was therefore selected for further development.

2.2.5 determination of the affinity of Fab fragments of the humanized variant of the murine CEA-antibody A5B7 for human CEA Using surface plasmon resonance (BIACORE)

The affinity of Fab fragments of the humanized variant of murine CEA antibody A5B7 for human CEA was assessed by surface plasmon resonance using a BIACORE T200 instrument. Human CEA (hu N (A2-B2) A-avi-His B) was immobilized onto flow cell 2 at a concentration of 40nM by standard amine coupling on a CM5 chip, for a duration of time30s, to about 100 RU. The Fab fragment of the humanized variant of murine CEA antibody A5B7 was then injected as analyte (3-fold dilution in a concentration range of 500-0.656nM) for a contact time of 120s, a dissociation time of 250s or 1000s, and a flow rate of 30. mu.l/min. Regeneration of human CEA (hu N (A2-B2) A-avi-His B) levels was achieved by 2 pulse injections of 10mM glycine/HCl (pH 2.0) over 60 s. The data were double referenced against the non-immobilized flow cell 1 and zero concentration analyte. Sensorgrams of the analytes were fitted to a simple 1:1Langmuir interaction model. Affinity constant [ K ] for human CEA (A2 Domain) _D]Summarized in table 15 below.

Table 15: fab fragments representing different humanized variants of murine CEA antibody A5B7 have affinity constants for human CEA (a2 domain).

The humanized variant of murine CEA antibody A5B7 has lower affinity than the parent murine antibody. The Fab fragment P1AE4138 (heavy chain comprising the VH variant 3-23A5-1A and Ckappa light chain comprising the VL variant A5-L1D) derived from P1AE2167 was selected as the final humanized variant. Furthermore, to remove the deamidation site, a glycine to alanine mutation at Kabat position 54(G54A) was introduced in the VH domain, resulting in VL variant 3-23a 5-1E. The final humanized antibody (heavy chain comprising the VH variant 3-23A5-1E and Ckappa light chain comprising the VL variant A5-L1D) was designated A5H1EL1D or huA5B 7.

2.2.6 formation and production of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to CEA (A5H1EL1D)

Bispecific 2+ 1H 2H anti-4-1 BB anti-CEA huIgG 1P 329GLAL antibody was produced as described in example 2.1 for the 2+1 anti-4-1 BB anti-CEA (A5B7) huIgG 1P 329GLAL antibody.

The amino acid sequences of the bispecific 4-1BB (20H4.9)/CEA (A5H1EL1D) P329GLALA IgG 12 +1(H2H) antibodies can be found in Table 16. Proteins were produced and purified as described in example 1.2.

Table 16: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (A5H1EL1D) human IgG 1P 329 GLAA antigen binding molecule (2+ 1H 2H)

2.3 formation of humanized variants of anti-CEA antibody MFE23

2.3.1 methods

anti-CEA antibody MFE23 is disclosed by, for example, M.K.Boehm et al (biochem.J.2000,346,519-528), and its structure can be found in PDB ID:11QOK in Protein Structure database (PDB) (www.rcsb.org, H.M.Berman et al, The Protein Data Bank, Nucleic Acids Research, 2000, 28, 235-242). The entries include heavy and light chain variable domain sequences. To identify a suitable human acceptor framework in the humanization process of the anti-CEA binding agent MFE23, a classical approach was taken, i.e., to find an acceptor framework with high sequence homology, to graft CDRs onto this framework, and to evaluate conceivable back-mutations. More specifically, the effect of each amino acid difference of the identified framework and the parent antibody on the structural integrity of the binding agent is judged, and back mutations towards the parent sequence are introduced as appropriate. Structural assessment Fv region homology models based on the parental antibody and its humanized versions were created by an internal antibody structural homology modeling tool, which was implemented using the Biovia Discovery Studio Environment version 4.5.

To improve the confidence of the back-mutation selection, we identified the closest murine homologous sequence from which the antibody was likely derived. We sought therefrom the location where extensive somatic hypermutation occurred during murine B-cell maturation in this antibody. These mutations may be important for introgression into humanized constructs.

2.3.2 selection of acceptor frameworks and Adaptation thereof

The acceptor framework was selected as shown in table 17 below:

table 17: acceptor framework

The CDR3 post-framework region was adapted from the human J element germline IGHJ4-01 for the heavy chain and the sequence was similar to the kappa J element IGKJ4-01 for the light chain. Based on structural considerations, back mutations were introduced at Kabat positions 71 and 93 of the heavy chain from the human acceptor framework to the amino acids in the parent binding agent. Based on the importance of framework mutations in the murine germline (resulting in the final mature MFE23 sequence), the Kabat residue 94 of the VH was changed back to the murine sequence.

To evaluate further improvement in affinity and/or stability of MFE23 sequences, we introgressed the following mutations in the light chain sequence: phe26Leu, Ser30Pro or Tyr, Leu78Val, as described in C.P. Graff et al (Protein Engineering, Design & Selection 2004,17(4), 293-304).

2.3.3 VH and VL domains of the resulting humanized CEA antibody

The VH domain of the resulting humanized CEA antibody can be seen in table 18 below, and the VL domain of the resulting humanized CEA antibody can be seen in table 19 below.

Table 18: amino acid sequence of VH Domain of humanized CEA antibody (based on human acceptor framework IGHV1-2-02)

Table 19: amino acid sequence of VL domain of humanized CEA antibody (based on human receptor framework IGKV1-39-01)

FIG. 8 shows an alignment of the sequences listed in tables 18 and 19, respectively.

The variable regions encoding the 6 heavy and 6 light chain DNA sequences of the humanized CEA binding agent were subcloned into frame with the constant heavy or constant light chain of human IgG1 containing the P239G, L234A and L235A mutations to eliminate binding to Fc γ receptors (WO 2012/130831 a 1). Antibodies were produced as follows. The resulting 36 variants (table 20) were tested for binding on MKN45 cells; and 7 variants were selected for further development.

Table 20: nomenclature of VH/VL combinations expressed as huIgG1_ LALA _ PG antibodies

MFE-L24

MFE-L25

MFE-L26

MFE-L27

MFE-L28

MFE-L29

MFE-H24

P1AE3125

P1AE3119

P1AE3113

P1AE3107

P1AE3101

P1AE3095

MFE-H25

P1AE3124

P1AE3118

P1AE3112

P1AE3106

P1AE3100

P1AE3094

MFE-H26

P1AE3123

P1AE3117

P1AE3111

P1AE3105

P1AE3099

P1AE3093

MFE-H27

P1AE3122

P1AE3116

P1AE3110

P1AE3104

P1AE3098

P1AE3092

MFE-H28

P1AE3121

P1AE3115

P1AE3109

P1AE3103

P1AE3097

P1AE3091

MFE-H29

P1AE3120

P1AE3114

P1AE3108

P1AE3102

P1AE3096

P1AE3090

2.3.4 selection of humanized MFE23 antibodies

Binding of 36 humanized MFE23 huIgG 1P 329G LALA variants to CEA expressed on MKN45 cells was compared to the corresponding parental murine MFE23 huIgG 1P 329G LALA antibody. 17 clones lost binding to MKN45 cells expressing human CEACAM5 (fig. 9A). Binding was reduced for 8 clones compared to parental clone MFE23 (fig. 9B). The binding capacity of 11 clones was similar compared to the parental clone MFE23 (fig. 9C). Fitting of these binding curves EC ₅₀The values and the area under the curve (AUC) are shown in table 21.

Table 21: EC for binding curves of different humanized MFE23 huIgG 1P 329G LALA antibodies₅₀And area under the curve (AUC) are shown in FIGS. 8A, 8B and 8C

2.3.5 formation and production of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to humanized CEA (MFE23) Binders

Bispecific 2+ 1H 2H anti-4-1 BB anti-CEA huIgG 1P 329GLAL antibody was produced as described in example 2.1 for the 2+1 anti-4-1 BB anti-CEA (MFE23) huIgG 1P 329GLAL antibody.

The amino acid sequences of bispecific 4-1BB (20H4.9)/CEA (humFE23-L28-H24) P329GLALA IgG12+1(H2H) antibodies can be found in Table 22, and the amino acid sequences of 4-1BB (20H4.9)/CEA (humFE23-L28-H28) P329GLALA IgG12+1(H2H) antibodies can be found in Table 23. Proteins were produced and purified as described in example 1.2. Other bispecific constructs are shown in tables 24 to 28.

Table 22: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (humFE23-L28-H24) human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H)

Table 23: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (humFE23-L28-H28) human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H)

Table 24: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (humFE23-L28-H25) human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H)

Table 25: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (humFE23-L27-H29) human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H)

Table 26: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (humFE23-L27-H28) human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H)

Table 27: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (humFE23-L27-H26) human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H)

Table 28: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CEA (humFE23-L27-H24) human IgG 1P 329GLALA antigen binding molecule (2+ 1H 2H)

Bispecific antibody production was performed as described in example 1.2. An exemplary analysis of the products obtained is shown in table 29 below.

TABLE 29 Biochemical analysis of anti-4-1 BB, anti-CEA huIgG1 PGLALA bispecific antibodies with 2+ 1H 2H

Example 3

Preparation, purification and characterization of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to CD19

2.1 formation and production of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to CD19

Bispecific agonistic 4-1BB antibodies with bivalent binding to 4-1BB and monovalent binding to CEA can also be prepared by substituting anti-FAP cross Fab for anti-CD 19 cross Fab. This construct is also known as the head-to-head (H2H)2+1 form.

The formation and preparation of the CD19 binding agent clone 2B11 is described in WO 2017/055328 a 1. The amino acid sequence of the 2+ 1H 2H bivalent bispecific 4-1BB (20H4.9) x CD19(2B11) P329GLALA IgG 12 +1(H2H) antibody huIgG1 PGLALA can be found in Table 30 below.

Table 30: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-CD 19(2B11) human IgG 1P 329GLALA antigen-binding molecule (2+ 1H 2H)

Example 4

Functional characterization of 2+ 1H 2H bispecific agonist 4-1BB antigen-binding molecules with monovalent binding to FAP

4.1 surface plasmon resonance (simultaneous coupling)

The ability to bind to both human 4-1BB Fc (kih) and human FAP was assessed using Surface Plasmon Resonance (SPR). All SPR experiments used Biacore T200 using HBS-EP as running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20, Biacore, Freiburg/Germany) at 25 ℃. Human 4-1BB Fc (kih) was coupled directly to the flow-through cell (CM5 sensor chip) by amine coupling. The immobilization level used was 710 Resonance Units (RU).

The 2+ 1H 2H anti-4-1 BB/anti FAP huIgG1 PGLALA construct was passed through the flow cell at a concentration of 200nM at a flow rate of 30 μ L/min for 90 seconds and dissociation was set to 0 seconds. Human FAP was injected as a second analyte into the flow-through cell at a concentration of 500nM at a flow rate of 30 μ L/min for 90 seconds (see assay setup in fig. 2A). Dissociation was monitored for 120 seconds. Bulk refractive index differences were corrected for by subtracting the responses obtained in the reference flow cell where the protein was not immobilized.

As can be seen in FIG. 2B, the 2+ 1H 2H 4-1BB (20H4.9)/FAP (4B9) P329GLALA IgG1 antibody construct binds to both human 4-1BB and human FAP.

4.2 performing a competition assay using tagLite to confirm bivalent binding to hu4-1BB

To confirm the bivalent binding of bispecific 2+ 1H 2H anti-4-1 BB, anti-FAP huIgG 1P 329GLALA antibody to hu4-1BB, competition assays were performed using time-resolved fluorescence resonance energy transfer (TR-FRET) (referred to as tagLite).

Binding of d 2-labeled 4-1BB (clone 20H4.9) IgG1 to Tb-labeled hu4-1BB-SNAP expressed on transfected Hek cells generated a TR-FRET signal. In a competition assay, the substitution of bound d 2-labeled 4-1BB (clone 20H4.9) IgG1 to unlabeled 2+ 1H 2H 4-1BB (20H4.9)/FAP (4B9) P329 GLAA IgG1 antibody (one of the Fab against 4-1BB has no free N-terminus) or unlabeled 2+1 VH/VL (C-terminus) 4-1BB (20H4.9)/FAP (4B9) P329 GLAA IgG1 antibody (two "free Fab" against 4-1 BB) resulted in a decrease in TR-FRET signal (Table 31).

Table 31: samples for use in competition assays

Briefly, pre-labeled Tb hu4-1BB-SNAP expressing cells were thawed, washed, and mixed at a density of 5000 cells per well (10. mu.L) with 5. mu.L of 4-1BB labeled with receptor (d2) (clone 20H4.9) IgG1 (concentration 0.6nM) and 5. mu.L of unlabeled competitor construct (1:3 dilution in the concentration range 0.006-1000 nM); the final volume in 384 well plates was 20 μ L. After 0h, 2h and 4h staining at RT, the fluorescence signal of the fluorescence donor (Tb) at 620nm and the fluorescence signal of the fluorescence acceptor (d2) at 665nm were measured (M1000 Pro, Tecan). Ratios of 665/620 × 10000(R) were calculated and the ratios of the reference (cells only) were subtracted to give plotted Δ R values. To determine IC ₅₀Single site-fitting log IC was used in Graph Pad Prism6₅₀The results were analyzed (table 32). The measurement was repeated twice.

Table 32: k after 4h_iValue and 95% confidence interval

The results indicate that both 4-1BB FAP bispecific constructs can compete with 4-1BB (clone 20H4.9) IgG for binding to hu4-1BB (IC)₅₀Similarly). This indicates that both Fab arms of the bispecific 2+1H2H anti-4-1 BB, anti-FAP huIgG 1P 329GLAL A antibody directed against 4-1BB can bind to 4-1 BB. Thus, 2+1H2H 4-1BB (20H4.9)/FThe AP (4B9) huIgG 1P 329GLALA antibody can bind bivalently to 4-1BB (FIG. 3).

4.3 binding to cell lines expressing human FAP

To test binding to cell surface expressed human Fibroblast Activation Protein (FAP), NIH/3T3-huFAP clone 19 cells were used. NIH/3T3-huFAP clone 19 was formed by transfecting mouse embryonic fibroblast NIH/3T3 cells (ATCC CRL-1658) with an expression pETR4921 plasmid encoding human FAP in the presence of a CMV promoter. The cells were maintained in DMEM (GIBCO provided by Life Technologies, Cat. No. 42340-. In the binding assay, 2X 10 will be used ⁵NIH/3T3-huFAP clone 19 cells of (9) were added to each well of a round bottom suspension cell 96-well plate (Greiner bio-one, cellstar, Cat. 650185). The cells were washed once with 200. mu.L of DPBS, and the pellet was then resuspended in 100. mu.L/well of cold DPBS buffer at 4 ℃ containing the immobilizeable reactive dye eFluor 450(eBioscience, Cat. No. 65086318) diluted 1: 5000. The well plates were incubated at 4 ℃ for 30 minutes and then washed once with 200 μ L of cold DPBS buffer at 4 ℃. Then, the cells were resuspended in 50 μ L/well of cold FACS buffer at 4 ℃ containing different titer concentrations of 2+ 1H 2H bispecific agonistic 4-1BB (20H4.9)/FAP (4B9) P329GLALA antibody (also known as anti-4-1 BB (20H4.9) x anti-FAP (4B9)2+ 1H 2H) or control molecule with monovalent binding to FAP, and then incubated at 4 ℃ in the dark for 1 hour. After four washes with 200. mu.L of DPBS/well, the cells were stained with 50. mu.L/well of 4 ℃ cold FACS buffer at 4 ℃ for 30 minutes, containing 2.5. mu.g/mL of PE-conjugated AffiniPure anti-human IgG Fc γ fragment-specific goat F (ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109-116-098). The cells were washed twice with 200. mu.L of 4 ℃ DPBS buffer and then resuspended in 50. mu.L/well 1% formaldehyde in DPBS for fixation. The same or next day cells were resuspended in 100. mu.L FACS buffer and MACSQurant was used Collection was performed by Analyzer 10(Miltenyi Biotec).

As shown in figure 4, FAP-targeting molecules bind efficiently to NIH/3T3-huFAP clone 19 cells expressing human FAP compared to hu IgG 1P 293G LALA versions of non-targeted FAP. Thus, the N-terminal fusion anti-FAP cross Fab (black filled circles and lines) has a higher affinity for FAP than the C-terminal fusion anti-FAP VH/VL domain (grey filled squares and dashed lines). This is reflected in the EC of the 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) huIgG 1P 329GLAL antibody₅₀Lower, and higher gMFI at saturation resulted in higher area under the curve (AUC) values. Fitting EC₅₀The values and area under the curve values are listed in table 33, and the fitted AUC values are listed in table 34.

Table 33: EC binding to FAP-expressing cell line NIH/3T3-huFAP clone 19₅₀Value of

Table 34: area under the Curve (AUC) values for binding to FAP expressing cell line NIH/3T3-huFAP clone 19

4.4 binding to the reporter cell line Jurkat-hu4-1BB-NF kappa B-luc2 expressing human 4-1BB

To determine binding to human 4-1BB (CD137) expressed on the cell surface, the Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 reporter cell line (Promega, Germany) was used. The cells were maintained as suspension cells in RPMI 1640 medium (GIBCO supplied by Life Technologies, Cat. No. 42401-042) supplemented with 10% (v/v) fetal bovine serum (FBS, GIBCO supplied by Life Technologies, Cat. No. 16000-044, Lot. No. 941273, gamma-irradiated, mycoplasma-free, heat-inactivated), 2mM L-alanyl-L-glutamine dipeptide (Gluta-M) AX-I, GIBCO supplied by Life Technologies, Cat. No. 35050-038), 1mM sodium pyruvate (SIGMA-Aldrich, Cat. No. S8636), and 1% (v/v) MEM-nonessential amino acid solution 100x (SIGMA-Aldrich, Cat. No. M7145), 600. mu.g/mL G-418(Roche, Cat. No. 04727894001), 400. mu.g/mL hygromycin B (Roche, Cat. No. 10843555001), and 25mM HEPES (Sigma Life science, Cat. No. H0887-100 mL). In the binding assay, 2X 10 will be used⁵Jurkat-hu4-1BB-NFkB-luc2 was added to each well of a round bottom suspension cell 96-well plate (Greiner bio-one, cell star, Cat. 650185). The cells were washed once with 200. mu.L of DPBS, and the pellet was then resuspended in 100. mu.L/well of cold DPBS buffer at 4 ℃ containing the immobilizeable reactive dye eFluor 450(eBioscience, Cat. No. 65086318) diluted 1: 5000. The well plates were incubated at 4 ℃ for 30 minutes and then washed once with 200 μ L of cold DPBS buffer at 4 ℃. Then, the cells were resuspended in 50 μ L/well of cold FACS buffer at 4 ℃ containing various titrated concentrations of 2+ 1H 2H agonist anti-4-1 BB (20H4.9) x anti FAP (4B9) huIgG 1P 329GLALA antibody or control molecule, and then incubated at 4 ℃ in the dark for 1 hour. After four washes with 200. mu.L of DPBS/well, the cells were stained with 50. mu.L/well of 4 ℃ cold FACS buffer at 4 ℃ for 30 minutes, containing 2.5. mu.g/mL of PE-conjugated AffiniPure anti-human IgG Fc γ fragment-specific goat F (ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109-116-098). The cells were washed twice with 200. mu.L of 4 ℃ DPBS buffer and then resuspended in 50. mu.L/well 1% formaldehyde in DPBS for fixation. Cells from the same or the next day were resuspended in 100. mu.L of FACS buffer and harvested using MACSQurant Analyzer X (Miltenyi Biotec).

As shown in FIG. 5, the 2+ 1H 2H agonistic anti-4-1 BB (20H4.9) x anti-FAP (4B9) huIgG 1P 329GLAL antibody bound 4-1BB similarly to its control anti-4-1 BB (20H4.9) huIgG 1P 329G LALA. Thus, N-terminal head-to-head fusion of anti-FAP cross fabs did not affect binding to 4-1 BB. EC of binding curve₅₀Values and AUC are listed in table 35 and table 36, respectively.

Table 35: EC of binding curves to cell-expressed human 4-1BB shown in FIG. 5₅₀Value summary

Table 36: a summary of AUC values for the binding curves to cell-expressed human 4-1BB shown in FIG. 5

4.5 NF-. kappa.B activation in the reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 expressing human 4-1BB and NF-. kappa.B-luciferase reporter genes

Agonistic binding of the 4-1BB (CD137) receptor to its ligand (4-1BBL) induces 4-1BB downstream signaling by activating nuclear factor kappa B (NFkB) and promotes survival and activity of CD 8T cells (Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS.4-1BB promoters the subvalval of CD8(+) T lymphocytes by encrypting expression of Bcl-x (L) and Bfl-1.J Immunol 2002; 169: 4882-. To monitor NF-. kappa.B activation mediated by 2+ 1H 2H anti-4-1 BB, anti-FAP huIgG1 PGLALA bispecific antibodies, the Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 reporter cell line was purchased from Promega (Germany). Cell culture methods were as described above (in combination with the reporter cell line Jurkat-hu4-1BB-NFkB-luc2 expressing human 4-1 BB). In the assay, cells were harvested and resuspended in assay medium RPMI 1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) GlutaMAX-I. 10 μ L of the extract containing 2X 10 ³Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 reporter cells were transferred to individual wells of a capped sterile white 384-well flat-bottomed tissue culture plate (Corning, Cat. No. 3826). Add 10 μ L of assay medium containing titrated concentrations of 2+ 1H 2H bispecific agonistic anti-4-1 BB (20H4.9) x anti-FAP (4B9) huIgG 1P 329GLALA antibody (also known as anti-4-1 BB (20H4.9) x anti-FAP (4B9)2+ 1H 2H) or control molecule with monovalent binding to FAP. Finally, 10. mu.L of assay medium alone or containing 1X 10⁴Culture media of FAP-expressing cells (human melanoma cell line WM-266-4(ATCC CRL-1676) or NIH/3T3-huFAP clone 19 (as described above), and the well plates were placed in a cell incubator at 37 ℃ and 5% CO₂Incubate for 6 hours. Add 6. mu.l fresh lysate to each wellFrozen One-Glo luciferase assay detection solution (Promega, catalog No. E6110) and luminescence intensity was measured immediately using a Tecan plate reader (integration time 500ms, no filter, signal acquisition at all wavelengths).

As shown in FIG. 6, in the absence of FAP-expressing cells, no molecule was able to induce strong activation of the human 4-1BB receptor in the Jurkat-hu4-1BB-NFkB-luc2 reporter cell line, resulting in NFkB activation and luciferase expression. In the presence of FAP-expressing cells such as WM-266-4 (human melanoma cell line, moderate FAP expression level) or NIH/3T3-huFAP clone 19 (human FAP transgenic mouse fibroblast cell line), cross-linking of bispecific 2+1 anti-4-1 BB x anti-FAP huIgG1 PGLALA antibody (2+ 1H 2H anti-4-1 BB (20H4.9) x anti-FAP (4B9) antibody, black filled circles and lines, or 2+1 VH/VL anti-4-1 BB (20H4.9) x anti-FAP (4B9) antibody, gray filled squares and dashed lines) resulted in a large increase in luciferase activity for NFkB activation in Jurkat-hu4-1BB-NFkB-luc2 reporter cell lines, above that mediated by non-targeted control anti-4-1 BB (20H4.9) x non-targeted (DP47)2+1 VH/VL (open gray squares and small dashed lines). Thus, bispecific 2+ 1H 2H anti-4-1 BB x anti-FAP huIgG 1P 329GLAL antibody (anti-4-1 BB (20H4.9) x anti-FAP (4B9)2+ 1H 2H, black filled circles and lines) showed slightly higher degree of activation (EC ₅₀Lower value), possibly reflecting a higher affinity for FAP. EC of activation Curve₅₀The values and area under the curve (AUC) are listed in tables 37 and 38.

Table 37: EC of the activation Curve shown in FIG. 6₅₀Value of

Table 38: area under the Curve (AUC) values for the activation Curve shown in FIG. 6

Example 5

Functional characterization of 2+ 1H 2H bispecific agonistic 4-1BB antigen-binding molecules with monovalent binding to CEA

5.1 surface plasmon resonance (simultaneous binding)

The ability to bind both human 4-1BB Fc (kih) in the form of a NABA construct and human CEA was assessed using Surface Plasmon Resonance (SPR). All SPR experiments used Biacore T200 using HBS-EP as running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20, Biacore, Freiburg/Germany) at 25 ℃. Human N (A2B2) a or (NA1) BA protein was coupled directly to the flow-through cell of the CM5 chip by amine coupling. The fixed level used was about 600 RU.

The CEA-targeting 4-1BB agonist construct was passed through the flow cell at a concentration of 200nM at a flow rate of 30 μ L/min for 90 seconds and dissociation was set to 0 seconds. Human 4-1BB Fc (kih) was injected as a second analyte at a concentration of 500nM at a flow rate of 30. mu.L/min into the flow-through cell over 90 seconds (FIG. 10A). Dissociation was monitored for 120 seconds. Bulk refractive index differences were corrected for by subtracting the responses obtained in the reference flow cell where the protein was not immobilized.

As can be seen in FIGS. 10B and 10C, 2+ 1H 2H 4-1BB (20H4.9)/CEA (A5B7) P329GLALA IgG1 and 2+ 1H 2H 4-1BB (20H4.9)/CEA (A5H1EL1D) huIgG 1P 329 GLAA can bind both human CEA (in the form of an N (A2B2) A construct) and human 4-1 BB. FIG. 10D shows 2+ 1H 2H 4-1BB (20H4.9)/CEA (MFE23) huIgG 1P 329GLAL A that binds both human CEA (in the form of a (NA1) BA construct) and human 4-1 BB.

5.2 binding to cell lines expressing Macaca fascicularis and human CEACAM5

A first cell line expressing cynomolgus monkey CEACAM5 or human CEACAM5 was formed. The full-length cDNA encoding human and cynomolgus CEACAM5 was subcloned into mammalian expression vectors. The plasmid was transfected into CHO-K1(ATCC CRL-9618) cells using Lipofectamine LTX reagent (Invitrogen, #15338100) according to the manufacturer's protocol. Stably transfected CEACAM5 positive CHO cells were maintained in DMEM/F-12 medium (GIBCO supplied by Life Technologies, #11320033) supplemented with 10% fetal bovine serum (FBS, GIBCO supplied by Life Technologies, Cat. No. 16000-044, batch No. 941273, gamma-irradiated, Mycoplasma-free, heat-inactivated) and 2mM L-alanyl-L-GlutamineAmide dipeptides (Gluta-MAX-I, GIBCO supplied by Life Technologies, Cat. No. 35050-. Two days after transfection, puromycin (Invivogen; # ant-pr-1) was added to a concentration of 6. mu.g/mL, and the cells were cultured for several generations. After primary screening, cells with high cell surface expression of human and cynomolgus monkey CEACAM5 were sorted using a BD FACSAria II cell sorter (BD Biosciences) (detection antibody anti-CD 66 clone cd66ab.1.1) and cultured to determine stable cell clones. Expression levels and stability were confirmed by flow cytometry analysis over 4 weeks. In the binding assay, CHO-k1-cynoCEACAM5 clone 8, CHO-k1-huCEACAM5 clone 11, CHO-k1-huCEACAM5 clone 12 or CHO-k1-huCEACAM5 clone 13 were harvested and washed with DPBS (GIBCO supplied by Life Technologies, # 14190-. The cells were washed and incubated at 3X 10 ⁴The density of cells/well was seeded in 384 well plates (Corning # 3830). The cells were centrifuged (350xg,5min), the supernatant removed, and the cells resuspended in 10 μ Ι/well of FACS buffer (DPBS, supplemented with 2% FBS, 5nM EDTA, 7.5mM sodium azide) containing either bispecific agonist 4-1BB (20H4.9) x CEA huIgG 1P 329GLALA antibody or control (starting concentration 300nM) titrated at 2+ 1H 2H. Cells were incubated at 4 ℃ for 30min and then washed twice with 80 μ L/well DPBS. Cells were resuspended in 10. mu.L/well of FACS buffer for 30 minutes at 4 ℃ containing 2.5. mu.g/mL of PE-conjugated AffiniPure anti-human IgG Fc γ -fragment specific goat F (ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109-116-098). Cells were washed twice with 80 μ L/well DPBS and then fixed in 30 μ L/well DPBS containing 1% formaldehyde for at least 15 minutes. Cells from the same or the next day were resuspended in 50 μ L wells of FACS buffer and harvested using macSQurant Analyzer X (Miltenyi Biotec).

As shown in fig. 12A to 12D, the 2+ 1H 2H bispecific agonistic 4-1BB (20H4.9) x CEA huIgG 1P 329 glaa antibody binds with high efficiency to CHO-k1 clone 12 and clone 13 cells expressing human CEACAM5 compared to the non-CEA targeted huIgG 1P 293G LALA form. In contrast, 2+ 1H 2H bispecific agonist only, 4-1BB (20H4.9) x C EA (A5B7) huIgG 1P 329GLAL antibody bound well to CHO-k1-cynoCEACAM5 cells expressing cynomolgus monkey CEACAM 5. The 2+ 1H 2H bispecific agonist 4-1BB (20H4.9) x CEA (A5H1EL1D) huIgG 1P 329GLALA antibody binds very weakly to CHO-k1-cynoCEACAM5 expressing cynomolgus monkey CECAM5, whereas the 2+ 1H 2H bispecific agonist 4-1BB (20H4.9) x CEA (MFE23) hu 1P 329GLALA antibody does not bind to CHO-k1-cynoCEACAM5 because the MFE23 clone is not human/cynomolgus monkey cross-reactive. Fitting EC₅₀The values and area under the curve values are listed in table 39, and the fitted AUC values are listed in table 40.

Table 39: EC binding to CEACAM5 expressing cell line shown in FIG. 9₅₀Value of

Table 40: FIG. 9 shows the area under the Curve (AUC) values for binding to a CEACAM5 expressing cell line

5.3 activation of NF-. kappa.B in the reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 expressing human 4-1BB and NF-. kappa.B-luciferase reporter genes

Agonistic binding of the 4-1BB (CD137) receptor to its ligand (4-1BBL) induces 4-1BB downstream signaling by activating nuclear factor kappa B (NF κ B) and promotes survival and activity of CD 8T cells (Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS.4-1BB promoters the subvalval of CD8(+) T lymphocytes by secreting expression of Bcl-x (L) and Bfl-1.J Immunol 2002; 169: 4882-. To monitor NF κ B activation mediated by 2+ 1H 2H anti-4-1 BB, anti-CEA huIgG1 PGLALA bispecific antibodies, the Jurkat-hu4-1BB-NF κ B-luc2 reporter cell line was purchased from Promega (Germany). The cell culture method is as described above. In the assay, cells were harvested and resuspended in assay medium RPMI 1640 medium supplemented with 10% (v/v) FBS and 1% (v/v) GlutaMAX-I. 10 μ L of the extract containing 2X 10 ³Transfer of Jurkat-hu4-1BB-NF kappa B-luc2 reporter cells toIn each well of a capped sterile white 384-well flat-bottom tissue culture plate (Corning, catalog No. 3826). 10 μ L of assay medium containing a titrated concentration of a 2+ 1H 2H bispecific agonistic anti-4-1 BB (20H4.9) x anti-CEA huIgG 1P 329GLALA antibody (different clones, e.g., A5B7, A5H1EL1D or MFE23) with monovalent binding to CEA or a control molecule was added. Finally, 10. mu.L of assay medium alone or containing 1X 10⁴Culture media of different CHO-k1 cells (transfected with cynomolgus monkey or human CEACAM 5) and the well plates were placed in a cell culture incubator at 37 ℃ and 5% CO₂Incubate for 6 hours. To each well was added 6 μ l of freshly thawed One-Glo luciferase assay detection solution (Promega, catalog No. E6110) and the luminescence intensity was measured immediately using a Tecan plate reader (integration time 500ms, no filter, signal acquisition at all wavelengths).

As shown in fig. 13A to 13D, in the absence of CEACAM 5-expressing cells, no molecule was able to induce strong activation of the human 4-1BB receptor in Jurkat-hu4-1BB-NFkB-luc2 reporter cell line, leading to NFkB activation and luciferase expression. In the presence of cells expressing human CEACAM5 (e.g., CHO-k 1-human CEACAM5 clone 11 and CHO-k 1-human CEACAM5 clone 12), the cross-linking of bispecific 2+1 anti-4-1 BB, anti-CEA huIgG1 PGLALA antibody (2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA (MFE23) antibody, black filled circles and dashed lines, or 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA (A5B7) antibody, black filled diamonds and lines, or 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA (A5H1EL 1) 1D, gray downward triangles) resulted in a large increase in the activity of NFkB 4-1BB-NFkB-luc2 reporter cell line, NFkB-activated luciferase activity mediated by open igg 329 (glb 1) and gray-H2H 1H-1H-luc-gluc 2 control. In the presence of CHO-k1-cynoCEACAM5 clone 8, only the 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA (A5B7) huIgG 1P 329 GLAA antibody (black filled diamonds and lines) and the 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA (A5H1EL1D) huIgG 1P 329 GLAA antibody (grey downward triangles) induced a dramatic increase in NFkB-activated luciferase activity in Jurkat-hu4-1BB-NFkB-luc2 reporter cell line, but the 2+ 1H 2H anti-4-1 BB (20H4.9) x anti-CEA (MFE23) huIgG 1P 329 GLAA antibody (black filled circles and dashed lines) did not have this induction, since E23 did not have human/MFcynomolgus cross-reactive binding agents.

EC of activation Curve₅₀The values and area under the curve (AUC) are listed in tables 41 and 42.

Table 41: EC of the activation Curve shown in FIG. 10₅₀Value of

Table 42: area under the Curve (AUC) values for the activation Curve shown in FIG. 10

Example 6

Preparation, purification and characterization of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to PD-L1

2.1 formation and production of bispecific antibodies with bivalent binding to 4-1BB and monovalent binding to PD-L1

Bispecific agonistic 4-1BB antibodies with bivalent binding to 4-1BB and monovalent binding to PD-L1 can also be prepared by substituting anti-FAP cross Fab for anti-PD-L1 cross Fab. This construct is also known as the head-to-head (H2H)2+1 form.

The first heavy chain HC1 of this construct consisted of: VHCH1 of anti-4-1 BB binding agent (clone 20H4.9) followed by Fc wells. The second heavy chain HC2 consisted of VLCH1 followed by VHCH1 and Fc-protuberant anti-PD-L1 binding agent (clone yw243.55.s70, in cross-Fab form) against 4-1BB (clone 20H4.9) and consisting of a cross Fab. The PD-L1 binder yw243.55.s70 is described in WO 2010/077634. For the 4-1BB binder, the VH and VL sequences of clone 20H4.9 were obtained according to US 7,288,638B 2 or US 7,659,384B 2. The two heavy chains combined to form a heterodimer, which includes one PD-L1 binding cross Fab and two 4-1BB binding fabs (fig. 1E). Another heterodimer that binds monovalent to 4-1BB is formed by a first heavy chain HC1 (followed by Fc wells) of VHCH1 containing an anti-4-1 BB binding agent (clone 20H4.9) and a second heavy chain HC2 (followed by Fc protrusions) of VLCH1 containing an anti-PD-L1 binding agent (clone yw243.55.s70, in crossed Fab form) (fig. 1F).

To improve correct pairing, the following mutations were introduced in the CH-CL of the anti-4-1 BB Fab molecule: E123R and Q124K in CL and K147E and K213E in CH 1. The second light chain LC2 of the anti-PD-L1 binding agent consisted of VHCL (cross Fab). Using a bulge-entry-hole technique, heterodimers were formed by introducing Y349C/T366S/L368A/Y407V mutations in the first heavy chain HC1 (Fc-hole heavy chain) and by introducing S354C/T366W mutations in the second heavy chain HC2 (Fc-bulge heavy chain).

Furthermore, Pro329Gly, Leu234Ala and Leu235Ala mutations were introduced into the constant regions of the protuberance and pore heavy chains to eliminate binding to Fc γ receptors according to the methods described in international patent application publication No. WO2012/130831a 1.

Bispecific 2+ 1H 2H anti-4-1 BB anti-PD-L1 huIgG 1P 329GLAL antibody was produced as described in example 1.2 for the 2+1 anti-4-1 BB anti-FAP huIgG 1P 329GLAL antibody.

The amino acid sequences of the bispecific 4-1BB (20H 4.9)/PD-L1P 329 GLAA IgG 12 +1(H2H) antibodies are shown in Table 43, while the amino acid sequences of the bispecific 4-1BB (20H 4.9)/PD-L1P 329 GLAA IgG 11 +1 antibodies are shown in Table 44.

Proteins were produced and purified as described in example 1.2.

Table 43: amino acid sequence of bispecific bivalent anti-4-1 BB/monovalent anti-PD-L1 human IgG 1P 329 GLAA antibody (2+ 1H 2H)

Table 44: amino acid sequence of bispecific monovalent anti-4-1 BB/monovalent anti-PD-L1 human IgG 1P 329GLALA antigen-binding molecule (1+1)

Bispecific antibody production was performed as described in example 1.2. An exemplary analysis of the products obtained is shown in table 45 below.

TABLE 45 Biochemical analysis of anti-4-1 BB, anti-PD-L1 huIgG1 PGLALA

Example 7

Functional characterization of 2+ 1H 2H bispecific agonist 4-1BB antigen binding molecules with monovalent binding to PD-L1

7.1 surface plasmon resonance (simultaneous coupling)

The ability to bind to both human 4-1BB Fc (kih) and human PD-L1 was assessed using Surface Plasmon Resonance (SPR). All SPR experiments used Biacore T200 using HBS-EP as running buffer (0.01M HEPES pH 7.4, 0.15M NaCl, 3mM EDTA, 0.005% surfactant P20, Biacore, Freiburg/Germany) at 25 ℃. Human 4-1BB-fc (kih) protein was coupled directly to the flow cell of a CM5 chip by amine coupling. The fixed level used was about 900 RU.

The PD-L1 targeted 4-1BB agonist construct was passed through the flow cell at a concentration of 150nM at a flow rate of 10 μ L/min for 90 seconds and dissociation was set to 0 seconds. Human PD-L1-Fc (recombinant human PD-L1/B7-H1 Fc chimeric protein, 156-B7-100: R & D Systems) was injected into the flow cell as a second analyte at a concentration of 200nM for 90 seconds at a flow rate of 30. mu.L/min (FIG. 14A). Dissociation was monitored for 240 seconds. Bulk refractive index differences were corrected for by subtracting the responses obtained in the reference flow cell where the protein was not immobilized.

As can be seen in FIGS. 14B and 14C, the 2+1H2H and the 1+ 14-1 BB (20H4.9)/PD-L1 human IgG1 PGLALA antibodies can bind to both human PD-L1 and human 4-1BB antibodies.

7.2 binding to a cell line expressing human PD-L1

A first cell line expressing human PD-L1 was formed. The full-length cDNA encoding human PD-L1 was subcloned into a mammalian expression vector. Plasmids were transfected into MKN45(DSMZ 409) cells using Lipofectamine LTX reagent (Invitrogen, #15338100) according to the manufacturer's protocol. Stably transfected PD-L1 positive PD-L1 cells were maintained in RPMI 1640 medium (GIBCO supplied by Life Technologies, catalog No. 42401-042) supplemented with 10% fetal bovine serum (FBS, GIBCO supplied by Life Technologies, catalog No. 16000-044, lot No. 941273, gamma irradiation, mycoplasma free, heat inactivation), 2mM L-alanyl-L-glutamine dipeptide (Gluta-MAX-I, GIBCO supplied by Life Technologies, catalog No. 35050-038) and optionally 200. mu.g/mL hygromycin B (Roche, catalog No. 10843555001) and 1.5. mu.g/mL puromycin (GIBCO supplied by Life Technologies, catalog No. A11138-02). In the binding assay, MKN45 cells and MKN45-huPD-L1 were harvested, washed with DPBS (GIBCO, #14190- & 136 supplied by Life Technologies), and stained in DPBS containing the immobilizable reactive dye eF450(eBioscience #65-0863-18) at 4 ℃ for 30 minutes. The cells were washed and incubated at 3X 10 ⁴The density of cells/well was seeded in 384 well plates (Corning # 3830). The cells were centrifuged (350xg,5min), the supernatant removed, and the cells resuspended in 10 μ L/well FACS buffer (DPBS, supplemented with 2% FBS, 5nM EDTA, 7.5mM sodium azide) containing titrated concentrations of 2+ 1H 2H bispecific agonist 4-1BB (20H4.9) x PD-L1huIgG 1P329glal, 1+1 bispecific agonist 4-1BB (20H4.9) x PD-L1huIgG 1P329glal antibody or control (starting concentration 300 nM). Cells were incubated at 4 ℃ for 30min and then washed twice with 80 μ L/well DPBS. Cells were resuspended in 10. mu.L/well of FACS buffer for 30 minutes at 4 ℃ containing 2.5. mu.g/mL of PE-conjugated AffiniPure anti-human IgG Fc γ -fragment specific goat F (ab')2 fragment (Jackson ImmunoResearch, Cat. No. 109-116-098). Cells were washed twice with 80 μ L/well DPBS and then fixed in 30 μ L/well DPBS containing 1% formaldehyde for at least 15 minutes. Cells from the same or the next day were resuspended in 50 μ L wells of FACS buffer and harvested using macSQurant Analyzer X (Miltenyi Biotec).

As shown in fig. 15B, the 2+ 1H 2H bispecific agonist 4-1BB (20H4.9) x PD-L1huIgG 1P329 glaa antibody (black triangles and lines) and 1+1 bispecific agonist 4-1BB (20H4.9) x PD-L1 (gray triangles and lines) bind to MKN45-huPD-L1 cells expressing human PD-L1 with high efficiency compared to the non-PD-L1 targeting huIgG1P 293 glaa format, but not to the parental cell line MKN 45. Fitting EC ₅₀The values and area under the curve values are listed in table 46, and the fitted AUC values are listed in table 47.

Table 46: EC binding to PD-L1 expressing cell line shown in FIG. 15B₅₀Value of

Table 47: FIG. 15B shows area under the Curve (AUC) values for binding to a cell line expressing PD-L1

7.3 NF-. kappa.B activation in the reporter cell line Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 expressing human 4-1BB and NF-. kappa.B-luciferase reporter genes

Agonistic binding of the 4-1BB (CD137) receptor to its ligand (4-1BBL) induces 4-1BB downstream signaling by activating nuclear factor kappa B (NFkB) and promotes survival and activity of CD 8T cells (Lee HW, Park SJ, Choi BK, Kim HH, Nam KO, Kwon BS.4-1BB promoters the subvalval of CD8(+) T lymphocytes by encrypting expression of Bcl-x (L) and Bfl-1.J Immunol 2002; 169: 4882-. To monitor NF-. kappa.B activation mediated by the 2+ 1H 2H anti-4-1 BB x anti-PD-L1 huIgG1 PGLALA bispecific antibody, a Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 reporter cell line was purchased from Promega (Germany). The cell culture method is as described above. In the assay, cells were harvested and resuspended in 10% (v/v) FBS supplemented with 1% (v/v) GlutaMAX-IAssay medium RPMI 1640 medium. 10 μ L of the extract containing 2X 10 ³Jurkat-hu4-1 BB-NF-. kappa.B-luc 2 reporter cells were transferred to individual wells of a capped sterile white 384-well flat-bottomed tissue culture plate (Corning, Cat. No. 3826). mu.L of assay medium containing a titrated concentration of 2+ 1H 2H bispecific agonistic anti-4-1 BB (20H4.9) x anti-PD-L1 huIgG 1P 329 GLAA antibody, 1+1 bispecific agonistic anti-4-1 BB (20H4.9) x anti-PD-L1 huIgG 1P 329 GLAA antibody or control molecule was added. Finally, 10. mu.L of assay medium alone or containing 1X 10⁴Assay medium for individual parental MKN45 or MKN45 cells (transfected with human PD-L1), and the well plates were placed in a cell culture incubator at 37 ℃ and 5% CO₂Incubate for 6 hours. To each well was added 6 μ l of freshly thawed One-Glo luciferase assay detection solution (Promega, catalog No. E6110) and the luminescence intensity was measured immediately using a Tecan plate reader (integration time 500ms, no filter, signal acquisition at all wavelengths).

As shown in figures 16A to 16C, in the absence of PD-L1 expressing cells, no molecule was able to induce strong activation of the human 4-1BB receptor in Jurkat-hu4-1BB-NFkB-luc2 reporter cell line, leading to NFkB activation and subsequent luciferase expression. Crosslinking of bispecific 2+ 1H 2H anti-4-1 BB, anti-PD-L1 huIgG1 PGLALA antibodies (black triangles and lines) or bispecific 1+1 anti-4-1 BB, anti-PD-L1 huIgG1 PGLALA antibodies (gray triangles and lines) in the presence of MKN45 cells expressing human PD-L1 resulted in a greatly increased luciferase activity for NFkB activation in Jurkat-hu4-1BB-NFkB-luc2 reporter cell lines, above that mediated by non-targeted control anti-4-1 BB (20H4.9) huIgG 1P 329GLALA (solid gray circles and lines). EC of activation Curve ₅₀The values and area under the curve (AUC) are listed in tables 48 and 49.

Table 48: EC of the activation Curve shown in FIG. 16B₅₀Value of

Table 49: area under the Curve (AUC) values for the activation Curve shown in FIG. 16B

***

Sequence listing

<110> Haofmii Roche GmbH (F. Hoffmann-La Roche AG)

<120> novel bispecific agonistic 4-1BB antigen-binding molecules

<130> P34875-WO

<150> EP18181652.1

<151> 2018-07-04

<160> 196

<170> PatentIn 3.5 edition

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB (20H4.9) CDR-H1

<400> 1

Gly Tyr Tyr Trp Ser

1 5

<210> 2

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB (20H4.9) CDR-H2

<400> 2

Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser

1 5 10 15

<210> 3

<211> 13

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB (20H4.9) CDR-H3

<400> 3

Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu

1 5 10

<210> 4

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB (20H4.9) CDR-L1

<400> 4

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB (20H4.9) CDR-L2

<400> 5

Asp Ala Ser Asn Arg Ala Thr

1 5

<210> 6

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB (20H4.9) CDR-L3

<400> 6

Gln Gln Arg Ser Asn Trp Pro Pro Ala Leu Thr

1 5 10

<210> 7

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB (20H4.9) VH

<400> 7

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 109

<212> PRT

<213> Artificial sequence

<220>

<223> 4-1BB (20H4.9) VL

<400> 8

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 9

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(4B9) CDR-H1

<400> 9

Ser Tyr Ala Met Ser

1 5

<210> 10

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(4B9) CDR-H2

<400> 10

Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val Lys

1 5 10 15

Gly

<210> 11

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(4B9) CDR-H3

<400> 11

Gly Trp Phe Gly Gly Phe Asn Tyr

1 5

<210> 12

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(4B9) CDR-L1

<400> 12

Arg Ala Ser Gln Ser Val Thr Ser Ser Tyr Leu Ala

1 5 10

<210> 13

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(4B9) CDR-L2

<400> 13

Val Gly Ser Arg Arg Ala Thr

1 5

<210> 14

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(4B9) CDR-L3

<400> 14

Gln Gln Gly Ile Met Leu Pro Pro Thr

1 5

<210> 15

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(28H1) CDR-H1

<400> 15

Ser His Ala Met Ser

1 5

<210> 16

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(28H1) CDR-H2

<400> 16

Ala Ile Trp Ala Ser Gly Glu Gln Tyr Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 17

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(28H1) CDR-H3

<400> 17

Gly Trp Leu Gly Asn Phe Asp Tyr

1 5

<210> 18

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(28H1) CDR-L1

<400> 18

Arg Ala Ser Gln Ser Val Ser Arg Ser Tyr Leu Ala

1 5 10

<210> 19

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(28H1) CDR-L2

<400> 19

Gly Ala Ser Thr Arg Ala Thr

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(28H1) CDR-L3

<400> 20

Gln Gln Gly Gln Val Ile Pro Pro Thr

1 5

<210> 21

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(4B9) VH

<400> 21

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(4B9) VL

<400> 22

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro

85 90 95

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 23

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(28H1) VH

<400> 23

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser His

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Trp Ala Ser Gly Glu Gln Tyr Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Gly Trp Leu Gly Asn Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 24

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> FAP(28H1) VL

<400> 24

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Arg Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Ile Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Gln Val Ile Pro

85 90 95

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 25

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-H1

<400> 25

Asp Tyr Tyr Met Asn

1 5

<210> 26

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-H2

<400> 26

Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 27

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-H3

<400> 27

Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-L1

<400> 28

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-L2

<400> 29

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 30

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7)- CDR-L3

<400> 30

Gln His Trp Ser Ser Lys Pro Pro Thr

1 5

<210> 31

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7) VH (parental)

<400> 31

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210> 32

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5B7) VL (parental)

<400> 32

Gln Thr Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 33

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-H1

<400> 33

Asp Ser Tyr Met His

1 5

<210> 34

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-H2

<400> 34

Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln

1 5 10 15

Gly

<210> 35

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-H3

<400> 35

Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr

1 5 10

<210> 36

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-L1

<400> 36

Ser Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 37

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-L2

<400> 37

Ser Thr Ser Asn Leu Ala Ser

1 5

<210> 38

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23)- CDR-L3

<400> 38

Gln Gln Arg Ser Ser Tyr Pro Leu Thr

1 5

<210> 39

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23) VH

<400> 39

Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Leu Arg Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 40

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE23) VL

<400> 40

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 41

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-H1

<400> 41

Asp Thr Tyr Met His

1 5

<210> 42

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-H2

<400> 42

Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe Gln

1 5 10 15

Gly

<210> 43

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-H3

<400> 43

Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr

1 5 10

<210> 44

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-L1

<400> 44

Arg Ala Gly Glu Ser Val Asp Ile Phe Gly Val Gly Phe Leu His

1 5 10 15

<210> 45

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-L2

<400> 45

Arg Ala Ser Asn Arg Ala Thr

1 5

<210> 46

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA)- CDR-L3

<400> 46

Gln Gln Thr Asn Glu Asp Pro Tyr Thr

1 5

<210> 47

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA) VH

<400> 47

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 48

<211> 111

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (T84.66-LCHA) VL

<400> 48

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 49

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CH1A1A 98/99)- CDR-H1

<400> 49

Glu Phe Gly Met Asn

1 5

<210> 50

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CH1A1A 98/99)- CDR-H2

<400> 50

Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe Lys

1 5 10 15

Gly

<210> 51

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CH1A1A 98/99)- CDR-H3

<400> 51

Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr

1 5 10

<210> 52

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CEA 2F1)- CDR-L1

<400> 52

Lys Ala Ser Ala Ala Val Gly Thr Tyr Val Ala

1 5 10

<210> 53

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CEA 2F1)- CDR-L2

<400> 53

Ser Ala Ser Tyr Arg Lys Arg

1 5

<210> 54

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CEA 2F1)- CDR-L3

<400> 54

His Gln Tyr Tyr Thr Tyr Pro Leu Phe Thr

1 5 10

<210> 55

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CH1A1A 98/99) VH

<400> 55

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 56

<211> 108

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (CEA 2F1) VL

<400> 56

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 57

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CD19 (8B8-2B11) CDR-H1

<400> 57

Asp Tyr Ile Met His

1 5

<210> 58

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CD19 (8B8-2B11) CDR-H2

<400> 58

Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe Gln

1 5 10 15

Gly

<210> 59

<211> 12

<212> PRT

<213> Artificial sequence

<220>

<223> CD19 (8B8-2B11) CDR-H3

<400> 59

Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr

1 5 10

<210> 60

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> CD19 (8B8-2B11) CDR-L1

<400> 60

Lys Ser Ser Gln Ser Leu Glu Thr Ser Thr Gly Thr Thr Tyr Leu Asn

1 5 10 15

<210> 61

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CD19 (8B8-2B11) CDR-L2

<400> 61

Arg Val Ser Lys Arg Phe Ser

1 5

<210> 62

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CD19 (8B8-2B11) CDR-L3

<400> 62

Leu Gln Leu Leu Glu Asp Pro Tyr Thr

1 5

<210> 63

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CD19 (8B8-2B11) VH

<400> 63

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 64

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> CD19 (8B8-2B11) VL

<400> 64

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser

20 25 30

Thr Gly Thr Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu

85 90 95

Leu Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 65

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> VHCH1(EE) (20H4.9) -heavy chain HC1 (Fc well)

<400> 65

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro

<210> 66

<211> 673

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (4B9) VHCH1(EE) (20H4.9) -heavy chain HC2 (Fc protrusions)

<400> 66

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Asn Val Gly Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Gly Ile Met Leu Pro

85 90 95

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

225 230 235 240

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

245 250 255

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

260 265 270

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

275 280 285

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

290 295 300

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

305 310 315 320

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

325 330 335

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

340 345 350

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

355 360 365

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

370 375 380

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

385 390 395 400

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

405 410 415

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

420 425 430

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

435 440 445

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

450 455 460

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

465 470 475 480

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

485 490 495

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

500 505 510

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

515 520 525

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

530 535 540

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

545 550 555 560

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

565 570 575

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

580 585 590

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

595 600 605

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

610 615 620

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

625 630 635 640

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

645 650 655

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

660 665 670

Pro

<210> 67

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> VLCL (RK) -light chain (20H4.9)

<400> 67

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 68

<211> 224

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (4B9)

<400> 68

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe

115 120 125

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

130 135 140

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

145 150 155 160

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

165 170 175

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

180 185 190

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

195 200 205

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 69

<211> 225

<212> PRT

<213> Artificial sequence

<220>

<223> Fc pore chain

<400> 69

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro

225

<210> 70

<211> 422

<212> PRT

<213> Artificial sequence

<220>

<223> human 4-1BB antigen Fc pro-chain

<400> 70

Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn

1 5 10 15

Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser

20 25 30

Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val

35 40 45

Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp

50 55 60

Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu

65 70 75 80

Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp

85 90 95

Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro

100 105 110

Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr

115 120 125

Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro

130 135 140

Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His

145 150 155 160

Ser Pro Gln Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser Pro Lys

165 170 175

Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

180 185 190

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

195 200 205

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

210 215 220

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

225 230 235 240

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

245 250 255

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

260 265 270

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

275 280 285

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

290 295 300

Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln

305 310 315 320

Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

325 330 335

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

340 345 350

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

355 360 365

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

370 375 380

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

385 390 395 400

Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln

405 410 415

Lys Ile Glu Trp His Glu

420

<210> 71

<211> 587

<212> PRT

<213> Artificial sequence

<220>

<223> VHCH1 (20H4.9) -heavy chain HC1 (Fc pore) -VH (4B9)

<400> 71

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly

465 470 475 480

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

485 490 495

Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly

500 505 510

Lys Gly Leu Glu Trp Val Ser Ala Ile Ile Gly Ser Gly Ala Ser Thr

515 520 525

Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

530 535 540

Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

545 550 555 560

Thr Ala Val Tyr Tyr Cys Ala Lys Gly Trp Phe Gly Gly Phe Asn Tyr

565 570 575

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

580 585

<210> 72

<211> 578

<212> PRT

<213> Artificial sequence

<220>

<223> VHCH1 (20H4.9) -heavy chain HC2 (Fc protrusions) -VL (4B9)

<400> 72

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr

465 470 475 480

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

485 490 495

Gln Ser Val Thr Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

500 505 510

Gln Ala Pro Arg Leu Leu Ile Asn Val Gly Ser Arg Arg Ala Thr Gly

515 520 525

Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

530 535 540

Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

545 550 555 560

Gln Gly Ile Met Leu Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu

565 570 575

Ile Lys

<210> 73

<211> 216

<212> PRT

<213> Artificial sequence

<220>

<223> VLCL-light chain (20H4.9)

<400> 73

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Ala Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 74

<211> 575

<212> PRT

<213> Artificial sequence

<220>

<223> VHCH1 (20H4.9) -heavy chain HC1 (Fc pore) -VH (DP47)

<400> 74

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

450 455 460

Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu

465 470 475 480

Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp

485 490 495

Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser

500 505 510

Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe

515 520 525

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn

530 535 540

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Ser

545 550 555 560

Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

565 570 575

<210> 75

<211> 578

<212> PRT

<213> Artificial sequence

<220>

<223> VHCH1 (20H4.9) -heavy chain HC2 (Fc protrusions) -VL (DP47)

<400> 75

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

100 105 110

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

450 455 460

Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr

465 470 475 480

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

485 490 495

Gln Ser Val Ser Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly

500 505 510

Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly

515 520 525

Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu

530 535 540

Thr Ile Ser Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

545 550 555 560

Gln Tyr Gly Ser Ser Pro Leu Thr Phe Gly Gln Gly Thr Lys Val Glu

565 570 575

Ile Lys

<210> 76

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (A5B7) VHCH1(EE) (20H4.9) -heavy chain HC2 (Fc protrusions)

<400> 76

Gln Thr Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 77

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (A5B7)

<400> 77

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Gln Ser Ile

65 70 75 80

Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Ser Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser

115 120 125

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

130 135 140

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

145 150 155 160

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

165 170 175

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

180 185 190

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

195 200 205

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

210 215 220

Arg Gly Glu Cys

225

<210> 78

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (MFE23) VHCH1(EE) (20H4.9) -heavy chain HC2 (Fc protrusions)

<400> 78

Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 79

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (MFE23)

<400> 79

Gln Val Lys Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Thr

1 5 10 15

Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Leu Arg Gln Gly Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Phe Thr Thr Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 80

<211> 676

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (T84.66-LCHA) VHCH1(EE) (20H4.9) -heavy chain HC2 (Fc protrusions)

<400> 80

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Glu Ser Val Asp Ile Phe

20 25 30

Gly Val Gly Phe Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Arg Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

115 120 125

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

180 185 190

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly

210 215 220

Gly Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys

225 230 235 240

Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe

245 250 255

Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu

260 265 270

Glu Trp Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro

275 280 285

Ser Leu Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln

290 295 300

Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr

305 310 315 320

Tyr Cys Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp

325 330 335

Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys

340 345 350

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

355 360 365

Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro

370 375 380

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

385 390 395 400

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

405 410 415

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

420 425 430

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro

435 440 445

Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

450 455 460

Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

465 470 475 480

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

485 490 495

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

500 505 510

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

515 520 525

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

530 535 540

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly

545 550 555 560

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

565 570 575

Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn

580 585 590

Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

595 600 605

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

610 615 620

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

625 630 635 640

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

645 650 655

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

660 665 670

Ser Leu Ser Pro

675

<210> 81

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (T84.66-LCHA)

<400> 81

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Ser Lys Tyr Val Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Pro Phe Gly Tyr Tyr Val Ser Asp Tyr Ala Met Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser

115 120 125

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

130 135 140

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

145 150 155 160

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

165 170 175

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

180 185 190

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

195 200 205

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

210 215 220

Arg Gly Glu Cys

225

<210> 82

<211> 673

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA 2F1) VHCH1(EE) (20H4.9) -heavy chain HC2 (Fc protrusions)

<400> 82

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Ala Ala Val Gly Thr Tyr

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Lys Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys His Gln Tyr Tyr Thr Tyr Pro Leu

85 90 95

Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

195 200 205

Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

210 215 220

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

225 230 235 240

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

245 250 255

Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile

260 265 270

Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu

275 280 285

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

290 295 300

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

305 310 315 320

Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly

325 330 335

Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

340 345 350

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

355 360 365

Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val

370 375 380

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

385 390 395 400

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

405 410 415

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

420 425 430

Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys

435 440 445

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

450 455 460

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

465 470 475 480

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

485 490 495

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

500 505 510

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

515 520 525

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

530 535 540

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile

545 550 555 560

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

565 570 575

Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

580 585 590

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

595 600 605

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

610 615 620

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

625 630 635 640

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

645 650 655

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

660 665 670

Pro

<210> 83

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA CH1A1A 98/99)

<400> 83

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Phe

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Thr Lys Thr Gly Glu Ala Thr Tyr Val Glu Glu Phe

50 55 60

Lys Gly Arg Val Thr Phe Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Asp Phe Ala Tyr Tyr Val Glu Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser

115 120 125

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

130 135 140

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

145 150 155 160

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

165 170 175

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

180 185 190

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

195 200 205

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

210 215 220

Arg Gly Glu Cys

225

<210> 84

<211> 677

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (2B11) VHCH1(EE) (20H4.9) -heavy chain HC2 (Fc protrusions)

<400> 84

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Glu Thr Ser

20 25 30

Thr Gly Thr Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Arg Val Ser Lys Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Leu

85 90 95

Leu Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu

145 150 155 160

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

165 170 175

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

180 185 190

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

195 200 205

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly

210 215 220

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu

225 230 235 240

Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser

245 250 255

Phe Ser Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly

260 265 270

Leu Glu Trp Ile Gly Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn

275 280 285

Pro Ser Leu Glu Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn

290 295 300

Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

305 310 315 320

Tyr Tyr Cys Ala Arg Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe

325 330 335

Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

340 345 350

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

355 360 365

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu

370 375 380

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

385 390 395 400

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

405 410 415

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

420 425 430

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu

435 440 445

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

450 455 460

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

465 470 475 480

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

485 490 495

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

500 505 510

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

515 520 525

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

530 535 540

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

545 550 555 560

Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

565 570 575

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

580 585 590

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

595 600 605

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

610 615 620

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

625 630 635 640

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

645 650 655

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

660 665 670

Leu Ser Leu Ser Pro

675

<210> 85

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (2B11)

<400> 85

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ile Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Ser Lys Tyr Thr Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ser Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Pro Gln Leu Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser

115 120 125

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

130 135 140

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

145 150 155 160

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

165 170 175

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

180 185 190

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

195 200 205

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

210 215 220

Arg Gly Glu Cys

225

<210> 86

<211> 760

<212> PRT

<213> Intelligent people

<400> 86

Met Lys Thr Trp Val Lys Ile Val Phe Gly Val Ala Thr Ser Ala Val

1 5 10 15

Leu Ala Leu Leu Val Met Cys Ile Val Leu Arg Pro Ser Arg Val His

20 25 30

Asn Ser Glu Glu Asn Thr Met Arg Ala Leu Thr Leu Lys Asp Ile Leu

35 40 45

Asn Gly Thr Phe Ser Tyr Lys Thr Phe Phe Pro Asn Trp Ile Ser Gly

50 55 60

Gln Glu Tyr Leu His Gln Ser Ala Asp Asn Asn Ile Val Leu Tyr Asn

65 70 75 80

Ile Glu Thr Gly Gln Ser Tyr Thr Ile Leu Ser Asn Arg Thr Met Lys

85 90 95

Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser Pro Asp Arg Gln Phe Val

100 105 110

Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp Arg Tyr Ser Tyr Thr Ala

115 120 125

Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly Glu Phe Val Arg Gly Asn

130 135 140

Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys Trp Ser Pro Val Gly Ser

145 150 155 160

Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile Tyr Leu Lys Gln Arg Pro

165 170 175

Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn Gly Arg Glu Asn Lys Ile

180 185 190

Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu Glu Glu Met Leu Ala Thr

195 200 205

Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly Lys Phe Leu Ala Tyr Ala

210 215 220

Glu Phe Asn Asp Thr Asp Ile Pro Val Ile Ala Tyr Ser Tyr Tyr Gly

225 230 235 240

Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile Pro Tyr Pro Lys Ala Gly

245 250 255

Ala Lys Asn Pro Val Val Arg Ile Phe Ile Ile Asp Thr Thr Tyr Pro

260 265 270

Ala Tyr Val Gly Pro Gln Glu Val Pro Val Pro Ala Met Ile Ala Ser

275 280 285

Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp Val Thr Asp Glu Arg Val

290 295 300

Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val Ser Val Leu Ser Ile

305 310 315 320

Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp Asp Cys Pro Lys Thr Gln

325 330 335

Glu His Ile Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe Phe Val

340 345 350

Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile Ser Tyr Tyr Lys Ile Phe

355 360 365

Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr Ile Lys Asp Thr Val

370 375 380

Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys Trp Glu Ala Ile Asn Ile

385 390 395 400

Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr Ser Ser Asn Glu Phe Glu

405 410 415

Glu Tyr Pro Gly Arg Arg Asn Ile Tyr Arg Ile Ser Ile Gly Ser Tyr

420 425 430

Pro Pro Ser Lys Lys Cys Val Thr Cys His Leu Arg Lys Glu Arg Cys

435 440 445

Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr Ala Lys Tyr Tyr Ala Leu

450 455 460

Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser Thr Leu His Asp Gly Arg

465 470 475 480

Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu Asn Lys Glu Leu Glu Asn

485 490 495

Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu Glu Ile Lys Lys Leu Glu

500 505 510

Val Asp Glu Ile Thr Leu Trp Tyr Lys Met Ile Leu Pro Pro Gln Phe

515 520 525

Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val Tyr Gly Gly Pro

530 535 540

Cys Ser Gln Ser Val Arg Ser Val Phe Ala Val Asn Trp Ile Ser Tyr

545 550 555 560

Leu Ala Ser Lys Glu Gly Met Val Ile Ala Leu Val Asp Gly Arg Gly

565 570 575

Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr Ala Val Tyr Arg Lys Leu

580 585 590

Gly Val Tyr Glu Val Glu Asp Gln Ile Thr Ala Val Arg Lys Phe Ile

595 600 605

Glu Met Gly Phe Ile Asp Glu Lys Arg Ile Ala Ile Trp Gly Trp Ser

610 615 620

Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu Ala Ser Gly Thr Gly Leu

625 630 635 640

Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp Glu Tyr Tyr

645 650 655

Ala Ser Val Tyr Thr Glu Arg Phe Met Gly Leu Pro Thr Lys Asp Asp

660 665 670

Asn Leu Glu His Tyr Lys Asn Ser Thr Val Met Ala Arg Ala Glu Tyr

675 680 685

Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly Thr Ala Asp Asp Asn

690 695 700

Val His Phe Gln Asn Ser Ala Gln Ile Ala Lys Ala Leu Val Asn Ala

705 710 715 720

Gln Val Asp Phe Gln Ala Met Trp Tyr Ser Asp Gln Asn His Gly Leu

725 730 735

Ser Gly Leu Ser Thr Asn His Leu Tyr Thr His Met Thr His Phe Leu

740 745 750

Lys Gln Cys Phe Ser Leu Ser Asp

755 760

<210> 87

<211> 748

<212> PRT

<213> Artificial sequence

<220>

<223> hu FAP extracellular domain + poly-lys-tag + his 6-tag

<400> 87

Arg Pro Ser Arg Val His Asn Ser Glu Glu Asn Thr Met Arg Ala Leu

1 5 10 15

Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr Lys Thr Phe Phe

20 25 30

Pro Asn Trp Ile Ser Gly Gln Glu Tyr Leu His Gln Ser Ala Asp Asn

35 40 45

Asn Ile Val Leu Tyr Asn Ile Glu Thr Gly Gln Ser Tyr Thr Ile Leu

50 55 60

Ser Asn Arg Thr Met Lys Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser

65 70 75 80

Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp

85 90 95

Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly

100 105 110

Glu Phe Val Arg Gly Asn Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys

115 120 125

Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile

130 135 140

Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn

145 150 155 160

Gly Arg Glu Asn Lys Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu

165 170 175

Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly

180 185 190

Lys Phe Leu Ala Tyr Ala Glu Phe Asn Asp Thr Asp Ile Pro Val Ile

195 200 205

Ala Tyr Ser Tyr Tyr Gly Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile

210 215 220

Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Val Val Arg Ile Phe Ile

225 230 235 240

Ile Asp Thr Thr Tyr Pro Ala Tyr Val Gly Pro Gln Glu Val Pro Val

245 250 255

Pro Ala Met Ile Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp

260 265 270

Val Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn

275 280 285

Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp

290 295 300

Asp Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp

305 310 315 320

Ala Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile

325 330 335

Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His

340 345 350

Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys

355 360 365

Trp Glu Ala Ile Asn Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr

370 375 380

Ser Ser Asn Glu Phe Glu Glu Tyr Pro Gly Arg Arg Asn Ile Tyr Arg

385 390 395 400

Ile Ser Ile Gly Ser Tyr Pro Pro Ser Lys Lys Cys Val Thr Cys His

405 410 415

Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr

420 425 430

Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser

435 440 445

Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu

450 455 460

Asn Lys Glu Leu Glu Asn Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu

465 470 475 480

Glu Ile Lys Lys Leu Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys Met

485 490 495

Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile

500 505 510

Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala

515 520 525

Val Asn Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala

530 535 540

Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr

545 550 555 560

Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr

565 570 575

Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile

580 585 590

Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu

595 600 605

Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val

610 615 620

Ser Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu Arg Phe Met Gly

625 630 635 640

Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val

645 650 655

Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His

660 665 670

Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala

675 680 685

Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser

690 695 700

Asp Gln Asn His Gly Leu Ser Gly Leu Ser Thr Asn His Leu Tyr Thr

705 710 715 720

His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp Gly Lys

725 730 735

Lys Lys Lys Lys Lys Gly His His His His His His

740 745

<210> 88

<211> 761

<212> PRT

<213> mouse

<400> 88

Met Lys Thr Trp Leu Lys Thr Val Phe Gly Val Thr Thr Leu Ala Ala

1 5 10 15

Leu Ala Leu Val Val Ile Cys Ile Val Leu Arg Pro Ser Arg Val Tyr

20 25 30

Lys Pro Glu Gly Asn Thr Lys Arg Ala Leu Thr Leu Lys Asp Ile Leu

35 40 45

Asn Gly Thr Phe Ser Tyr Lys Thr Tyr Phe Pro Asn Trp Ile Ser Glu

50 55 60

Gln Glu Tyr Leu His Gln Ser Glu Asp Asp Asn Ile Val Phe Tyr Asn

65 70 75 80

Ile Glu Thr Arg Glu Ser Tyr Ile Ile Leu Ser Asn Ser Thr Met Lys

85 90 95

Ser Val Asn Ala Thr Asp Tyr Gly Leu Ser Pro Asp Arg Gln Phe Val

100 105 110

Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp Arg Tyr Ser Tyr Thr Ala

115 120 125

Thr Tyr Tyr Ile Tyr Asp Leu Gln Asn Gly Glu Phe Val Arg Gly Tyr

130 135 140

Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys Trp Ser Pro Val Gly Ser

145 150 155 160

Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile Tyr Leu Lys Gln Arg Pro

165 170 175

Gly Asp Pro Pro Phe Gln Ile Thr Tyr Thr Gly Arg Glu Asn Arg Ile

180 185 190

Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu Glu Glu Met Leu Ala Thr

195 200 205

Lys Tyr Ala Leu Trp Trp Ser Pro Asp Gly Lys Phe Leu Ala Tyr Val

210 215 220

Glu Phe Asn Asp Ser Asp Ile Pro Ile Ile Ala Tyr Ser Tyr Tyr Gly

225 230 235 240

Asp Gly Gln Tyr Pro Arg Thr Ile Asn Ile Pro Tyr Pro Lys Ala Gly

245 250 255

Ala Lys Asn Pro Val Val Arg Val Phe Ile Val Asp Thr Thr Tyr Pro

260 265 270

His His Val Gly Pro Met Glu Val Pro Val Pro Glu Met Ile Ala Ser

275 280 285

Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp Val Ser Ser Glu Arg Val

290 295 300

Cys Leu Gln Trp Leu Lys Arg Val Gln Asn Val Ser Val Leu Ser Ile

305 310 315 320

Cys Asp Phe Arg Glu Asp Trp His Ala Trp Glu Cys Pro Lys Asn Gln

325 330 335

Glu His Val Glu Glu Ser Arg Thr Gly Trp Ala Gly Gly Phe Phe Val

340 345 350

Ser Thr Pro Ala Phe Ser Gln Asp Ala Thr Ser Tyr Tyr Lys Ile Phe

355 360 365

Ser Asp Lys Asp Gly Tyr Lys His Ile His Tyr Ile Lys Asp Thr Val

370 375 380

Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys Trp Glu Ala Ile Tyr Ile

385 390 395 400

Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr Ser Ser Asn Glu Phe Glu

405 410 415

Gly Tyr Pro Gly Arg Arg Asn Ile Tyr Arg Ile Ser Ile Gly Asn Ser

420 425 430

Pro Pro Ser Lys Lys Cys Val Thr Cys His Leu Arg Lys Glu Arg Cys

435 440 445

Gln Tyr Tyr Thr Ala Ser Phe Ser Tyr Lys Ala Lys Tyr Tyr Ala Leu

450 455 460

Val Cys Tyr Gly Pro Gly Leu Pro Ile Ser Thr Leu His Asp Gly Arg

465 470 475 480

Thr Asp Gln Glu Ile Gln Val Leu Glu Glu Asn Lys Glu Leu Glu Asn

485 490 495

Ser Leu Arg Asn Ile Gln Leu Pro Lys Val Glu Ile Lys Lys Leu Lys

500 505 510

Asp Gly Gly Leu Thr Phe Trp Tyr Lys Met Ile Leu Pro Pro Gln Phe

515 520 525

Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile Gln Val Tyr Gly Gly Pro

530 535 540

Cys Ser Gln Ser Val Lys Ser Val Phe Ala Val Asn Trp Ile Thr Tyr

545 550 555 560

Leu Ala Ser Lys Glu Gly Ile Val Ile Ala Leu Val Asp Gly Arg Gly

565 570 575

Thr Ala Phe Gln Gly Asp Lys Phe Leu His Ala Val Tyr Arg Lys Leu

580 585 590

Gly Val Tyr Glu Val Glu Asp Gln Leu Thr Ala Val Arg Lys Phe Ile

595 600 605

Glu Met Gly Phe Ile Asp Glu Glu Arg Ile Ala Ile Trp Gly Trp Ser

610 615 620

Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu Ala Ser Gly Thr Gly Leu

625 630 635 640

Phe Lys Cys Gly Ile Ala Val Ala Pro Val Ser Ser Trp Glu Tyr Tyr

645 650 655

Ala Ser Ile Tyr Ser Glu Arg Phe Met Gly Leu Pro Thr Lys Asp Asp

660 665 670

Asn Leu Glu His Tyr Lys Asn Ser Thr Val Met Ala Arg Ala Glu Tyr

675 680 685

Phe Arg Asn Val Asp Tyr Leu Leu Ile His Gly Thr Ala Asp Asp Asn

690 695 700

Val His Phe Gln Asn Ser Ala Gln Ile Ala Lys Ala Leu Val Asn Ala

705 710 715 720

Gln Val Asp Phe Gln Ala Met Trp Tyr Ser Asp Gln Asn His Gly Ile

725 730 735

Ser Ser Gly Arg Ser Gln Asn His Leu Tyr Thr His Met Thr His Phe

740 745 750

Leu Lys Gln Cys Phe Ser Leu Ser Asp

755 760

<210> 89

<211> 749

<212> PRT

<213> Artificial sequence

<220>

<223> murine FAP extracellular domain + poly-lys-tag + his 6-tag

<400> 89

Arg Pro Ser Arg Val Tyr Lys Pro Glu Gly Asn Thr Lys Arg Ala Leu

1 5 10 15

Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr Lys Thr Tyr Phe

20 25 30

Pro Asn Trp Ile Ser Glu Gln Glu Tyr Leu His Gln Ser Glu Asp Asp

35 40 45

Asn Ile Val Phe Tyr Asn Ile Glu Thr Arg Glu Ser Tyr Ile Ile Leu

50 55 60

Ser Asn Ser Thr Met Lys Ser Val Asn Ala Thr Asp Tyr Gly Leu Ser

65 70 75 80

Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp

85 90 95

Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Gln Asn Gly

100 105 110

Glu Phe Val Arg Gly Tyr Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys

115 120 125

Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile

130 135 140

Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Tyr Thr

145 150 155 160

Gly Arg Glu Asn Arg Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu

165 170 175

Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asp Gly

180 185 190

Lys Phe Leu Ala Tyr Val Glu Phe Asn Asp Ser Asp Ile Pro Ile Ile

195 200 205

Ala Tyr Ser Tyr Tyr Gly Asp Gly Gln Tyr Pro Arg Thr Ile Asn Ile

210 215 220

Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Val Val Arg Val Phe Ile

225 230 235 240

Val Asp Thr Thr Tyr Pro His His Val Gly Pro Met Glu Val Pro Val

245 250 255

Pro Glu Met Ile Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp

260 265 270

Val Ser Ser Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn

275 280 285

Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp His Ala Trp

290 295 300

Glu Cys Pro Lys Asn Gln Glu His Val Glu Glu Ser Arg Thr Gly Trp

305 310 315 320

Ala Gly Gly Phe Phe Val Ser Thr Pro Ala Phe Ser Gln Asp Ala Thr

325 330 335

Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His

340 345 350

Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys

355 360 365

Trp Glu Ala Ile Tyr Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr

370 375 380

Ser Ser Asn Glu Phe Glu Gly Tyr Pro Gly Arg Arg Asn Ile Tyr Arg

385 390 395 400

Ile Ser Ile Gly Asn Ser Pro Pro Ser Lys Lys Cys Val Thr Cys His

405 410 415

Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Tyr Lys

420 425 430

Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly Leu Pro Ile Ser

435 440 445

Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Gln Val Leu Glu Glu

450 455 460

Asn Lys Glu Leu Glu Asn Ser Leu Arg Asn Ile Gln Leu Pro Lys Val

465 470 475 480

Glu Ile Lys Lys Leu Lys Asp Gly Gly Leu Thr Phe Trp Tyr Lys Met

485 490 495

Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile

500 505 510

Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Lys Ser Val Phe Ala

515 520 525

Val Asn Trp Ile Thr Tyr Leu Ala Ser Lys Glu Gly Ile Val Ile Ala

530 535 540

Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Phe Leu His

545 550 555 560

Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Leu Thr

565 570 575

Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Glu Arg Ile

580 585 590

Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu

595 600 605

Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val

610 615 620

Ser Ser Trp Glu Tyr Tyr Ala Ser Ile Tyr Ser Glu Arg Phe Met Gly

625 630 635 640

Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val

645 650 655

Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His

660 665 670

Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala

675 680 685

Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser

690 695 700

Asp Gln Asn His Gly Ile Leu Ser Gly Arg Ser Gln Asn His Leu Tyr

705 710 715 720

Thr His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp Gly

725 730 735

Lys Lys Lys Lys Lys Lys Gly His His His His His His

740 745

<210> 90

<211> 748

<212> PRT

<213> Artificial sequence

<220>

<223> cynomolgus FAP extracellular domain + poly-lys-tag + his 6-tag

<400> 90

Arg Pro Pro Arg Val His Asn Ser Glu Glu Asn Thr Met Arg Ala Leu

1 5 10 15

Thr Leu Lys Asp Ile Leu Asn Gly Thr Phe Ser Tyr Lys Thr Phe Phe

20 25 30

Pro Asn Trp Ile Ser Gly Gln Glu Tyr Leu His Gln Ser Ala Asp Asn

35 40 45

Asn Ile Val Leu Tyr Asn Ile Glu Thr Gly Gln Ser Tyr Thr Ile Leu

50 55 60

Ser Asn Arg Thr Met Lys Ser Val Asn Ala Ser Asn Tyr Gly Leu Ser

65 70 75 80

Pro Asp Arg Gln Phe Val Tyr Leu Glu Ser Asp Tyr Ser Lys Leu Trp

85 90 95

Arg Tyr Ser Tyr Thr Ala Thr Tyr Tyr Ile Tyr Asp Leu Ser Asn Gly

100 105 110

Glu Phe Val Arg Gly Asn Glu Leu Pro Arg Pro Ile Gln Tyr Leu Cys

115 120 125

Trp Ser Pro Val Gly Ser Lys Leu Ala Tyr Val Tyr Gln Asn Asn Ile

130 135 140

Tyr Leu Lys Gln Arg Pro Gly Asp Pro Pro Phe Gln Ile Thr Phe Asn

145 150 155 160

Gly Arg Glu Asn Lys Ile Phe Asn Gly Ile Pro Asp Trp Val Tyr Glu

165 170 175

Glu Glu Met Leu Ala Thr Lys Tyr Ala Leu Trp Trp Ser Pro Asn Gly

180 185 190

Lys Phe Leu Ala Tyr Ala Glu Phe Asn Asp Thr Asp Ile Pro Val Ile

195 200 205

Ala Tyr Ser Tyr Tyr Gly Asp Glu Gln Tyr Pro Arg Thr Ile Asn Ile

210 215 220

Pro Tyr Pro Lys Ala Gly Ala Lys Asn Pro Phe Val Arg Ile Phe Ile

225 230 235 240

Ile Asp Thr Thr Tyr Pro Ala Tyr Val Gly Pro Gln Glu Val Pro Val

245 250 255

Pro Ala Met Ile Ala Ser Ser Asp Tyr Tyr Phe Ser Trp Leu Thr Trp

260 265 270

Val Thr Asp Glu Arg Val Cys Leu Gln Trp Leu Lys Arg Val Gln Asn

275 280 285

Val Ser Val Leu Ser Ile Cys Asp Phe Arg Glu Asp Trp Gln Thr Trp

290 295 300

Asp Cys Pro Lys Thr Gln Glu His Ile Glu Glu Ser Arg Thr Gly Trp

305 310 315 320

Ala Gly Gly Phe Phe Val Ser Thr Pro Val Phe Ser Tyr Asp Ala Ile

325 330 335

Ser Tyr Tyr Lys Ile Phe Ser Asp Lys Asp Gly Tyr Lys His Ile His

340 345 350

Tyr Ile Lys Asp Thr Val Glu Asn Ala Ile Gln Ile Thr Ser Gly Lys

355 360 365

Trp Glu Ala Ile Asn Ile Phe Arg Val Thr Gln Asp Ser Leu Phe Tyr

370 375 380

Ser Ser Asn Glu Phe Glu Asp Tyr Pro Gly Arg Arg Asn Ile Tyr Arg

385 390 395 400

Ile Ser Ile Gly Ser Tyr Pro Pro Ser Lys Lys Cys Val Thr Cys His

405 410 415

Leu Arg Lys Glu Arg Cys Gln Tyr Tyr Thr Ala Ser Phe Ser Asp Tyr

420 425 430

Ala Lys Tyr Tyr Ala Leu Val Cys Tyr Gly Pro Gly Ile Pro Ile Ser

435 440 445

Thr Leu His Asp Gly Arg Thr Asp Gln Glu Ile Lys Ile Leu Glu Glu

450 455 460

Asn Lys Glu Leu Glu Asn Ala Leu Lys Asn Ile Gln Leu Pro Lys Glu

465 470 475 480

Glu Ile Lys Lys Leu Glu Val Asp Glu Ile Thr Leu Trp Tyr Lys Met

485 490 495

Ile Leu Pro Pro Gln Phe Asp Arg Ser Lys Lys Tyr Pro Leu Leu Ile

500 505 510

Gln Val Tyr Gly Gly Pro Cys Ser Gln Ser Val Arg Ser Val Phe Ala

515 520 525

Val Asn Trp Ile Ser Tyr Leu Ala Ser Lys Glu Gly Met Val Ile Ala

530 535 540

Leu Val Asp Gly Arg Gly Thr Ala Phe Gln Gly Asp Lys Leu Leu Tyr

545 550 555 560

Ala Val Tyr Arg Lys Leu Gly Val Tyr Glu Val Glu Asp Gln Ile Thr

565 570 575

Ala Val Arg Lys Phe Ile Glu Met Gly Phe Ile Asp Glu Lys Arg Ile

580 585 590

Ala Ile Trp Gly Trp Ser Tyr Gly Gly Tyr Val Ser Ser Leu Ala Leu

595 600 605

Ala Ser Gly Thr Gly Leu Phe Lys Cys Gly Ile Ala Val Ala Pro Val

610 615 620

Ser Ser Trp Glu Tyr Tyr Ala Ser Val Tyr Thr Glu Arg Phe Met Gly

625 630 635 640

Leu Pro Thr Lys Asp Asp Asn Leu Glu His Tyr Lys Asn Ser Thr Val

645 650 655

Met Ala Arg Ala Glu Tyr Phe Arg Asn Val Asp Tyr Leu Leu Ile His

660 665 670

Gly Thr Ala Asp Asp Asn Val His Phe Gln Asn Ser Ala Gln Ile Ala

675 680 685

Lys Ala Leu Val Asn Ala Gln Val Asp Phe Gln Ala Met Trp Tyr Ser

690 695 700

Asp Gln Asn His Gly Leu Ser Gly Leu Ser Thr Asn His Leu Tyr Thr

705 710 715 720

His Met Thr His Phe Leu Lys Gln Cys Phe Ser Leu Ser Asp Gly Lys

725 730 735

Lys Lys Lys Lys Lys Gly His His His His His His

740 745

<210> 91

<211> 702

<212> PRT

<213> Intelligent people

<400> 91

Met Glu Ser Pro Ser Ala Pro Pro His Arg Trp Cys Ile Pro Trp Gln

1 5 10 15

Arg Leu Leu Leu Thr Ala Ser Leu Leu Thr Phe Trp Asn Pro Pro Thr

20 25 30

Thr Ala Lys Leu Thr Ile Glu Ser Thr Pro Phe Asn Val Ala Glu Gly

35 40 45

Lys Glu Val Leu Leu Leu Val His Asn Leu Pro Gln His Leu Phe Gly

50 55 60

Tyr Ser Trp Tyr Lys Gly Glu Arg Val Asp Gly Asn Arg Gln Ile Ile

65 70 75 80

Gly Tyr Val Ile Gly Thr Gln Gln Ala Thr Pro Gly Pro Ala Tyr Ser

85 90 95

Gly Arg Glu Ile Ile Tyr Pro Asn Ala Ser Leu Leu Ile Gln Asn Ile

100 105 110

Ile Gln Asn Asp Thr Gly Phe Tyr Thr Leu His Val Ile Lys Ser Asp

115 120 125

Leu Val Asn Glu Glu Ala Thr Gly Gln Phe Arg Val Tyr Pro Glu Leu

130 135 140

Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro Val Glu Asp Lys

145 150 155 160

Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Thr Gln Asp Ala Thr Tyr

165 170 175

Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg Leu Gln

180 185 190

Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn Val Thr Arg Asn

195 200 205

Asp Thr Ala Ser Tyr Lys Cys Glu Thr Gln Asn Pro Val Ser Ala Arg

210 215 220

Arg Ser Asp Ser Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Ala Pro

225 230 235 240

Thr Ile Ser Pro Leu Asn Thr Ser Tyr Arg Ser Gly Glu Asn Leu Asn

245 250 255

Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser Trp Phe

260 265 270

Val Asn Gly Thr Phe Gln Gln Ser Thr Gln Glu Leu Phe Ile Pro Asn

275 280 285

Ile Thr Val Asn Asn Ser Gly Ser Tyr Thr Cys Gln Ala His Asn Ser

290 295 300

Asp Thr Gly Leu Asn Arg Thr Thr Val Thr Thr Ile Thr Val Tyr Ala

305 310 315 320

Glu Pro Pro Lys Pro Phe Ile Thr Ser Asn Asn Ser Asn Pro Val Glu

325 330 335

Asp Glu Asp Ala Val Ala Leu Thr Cys Glu Pro Glu Ile Gln Asn Thr

340 345 350

Thr Tyr Leu Trp Trp Val Asn Asn Gln Ser Leu Pro Val Ser Pro Arg

355 360 365

Leu Gln Leu Ser Asn Asp Asn Arg Thr Leu Thr Leu Leu Ser Val Thr

370 375 380

Arg Asn Asp Val Gly Pro Tyr Glu Cys Gly Ile Gln Asn Lys Leu Ser

385 390 395 400

Val Asp His Ser Asp Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp

405 410 415

Asp Pro Thr Ile Ser Pro Ser Tyr Thr Tyr Tyr Arg Pro Gly Val Asn

420 425 430

Leu Ser Leu Ser Cys His Ala Ala Ser Asn Pro Pro Ala Gln Tyr Ser

435 440 445

Trp Leu Ile Asp Gly Asn Ile Gln Gln His Thr Gln Glu Leu Phe Ile

450 455 460

Ser Asn Ile Thr Glu Lys Asn Ser Gly Leu Tyr Thr Cys Gln Ala Asn

465 470 475 480

Asn Ser Ala Ser Gly His Ser Arg Thr Thr Val Lys Thr Ile Thr Val

485 490 495

Ser Ala Glu Leu Pro Lys Pro Ser Ile Ser Ser Asn Asn Ser Lys Pro

500 505 510

Val Glu Asp Lys Asp Ala Val Ala Phe Thr Cys Glu Pro Glu Ala Gln

515 520 525

Asn Thr Thr Tyr Leu Trp Trp Val Asn Gly Gln Ser Leu Pro Val Ser

530 535 540

Pro Arg Leu Gln Leu Ser Asn Gly Asn Arg Thr Leu Thr Leu Phe Asn

545 550 555 560

Val Thr Arg Asn Asp Ala Arg Ala Tyr Val Cys Gly Ile Gln Asn Ser

565 570 575

Val Ser Ala Asn Arg Ser Asp Pro Val Thr Leu Asp Val Leu Tyr Gly

580 585 590

Pro Asp Thr Pro Ile Ile Ser Pro Pro Asp Ser Ser Tyr Leu Ser Gly

595 600 605

Ala Asn Leu Asn Leu Ser Cys His Ser Ala Ser Asn Pro Ser Pro Gln

610 615 620

Tyr Ser Trp Arg Ile Asn Gly Ile Pro Gln Gln His Thr Gln Val Leu

625 630 635 640

Phe Ile Ala Lys Ile Thr Pro Asn Asn Asn Gly Thr Tyr Ala Cys Phe

645 650 655

Val Ser Asn Leu Ala Thr Gly Arg Asn Asn Ser Ile Val Lys Ser Ile

660 665 670

Thr Val Ser Ala Ser Gly Thr Ser Pro Gly Leu Ser Ala Gly Ala Thr

675 680 685

Val Gly Ile Met Ile Gly Val Leu Val Gly Val Ala Leu Ile

690 695 700

<210> 92

<211> 2322

<212> PRT

<213> Intelligent people

<400> 92

Met Gln Ser Gly Pro Arg Pro Pro Leu Pro Ala Pro Gly Leu Ala Leu

1 5 10 15

Ala Leu Thr Leu Thr Met Leu Ala Arg Leu Ala Ser Ala Ala Ser Phe

20 25 30

Phe Gly Glu Asn His Leu Glu Val Pro Val Ala Thr Ala Leu Thr Asp

35 40 45

Ile Asp Leu Gln Leu Gln Phe Ser Thr Ser Gln Pro Glu Ala Leu Leu

50 55 60

Leu Leu Ala Ala Gly Pro Ala Asp His Leu Leu Leu Gln Leu Tyr Ser

65 70 75 80

Gly Arg Leu Gln Val Arg Leu Val Leu Gly Gln Glu Glu Leu Arg Leu

85 90 95

Gln Thr Pro Ala Glu Thr Leu Leu Ser Asp Ser Ile Pro His Thr Val

100 105 110

Val Leu Thr Val Val Glu Gly Trp Ala Thr Leu Ser Val Asp Gly Phe

115 120 125

Leu Asn Ala Ser Ser Ala Val Pro Gly Ala Pro Leu Glu Val Pro Tyr

130 135 140

Gly Leu Phe Val Gly Gly Thr Gly Thr Leu Gly Leu Pro Tyr Leu Arg

145 150 155 160

Gly Thr Ser Arg Pro Leu Arg Gly Cys Leu His Ala Ala Thr Leu Asn

165 170 175

Gly Arg Ser Leu Leu Arg Pro Leu Thr Pro Asp Val His Glu Gly Cys

180 185 190

Ala Glu Glu Phe Ser Ala Ser Asp Asp Val Ala Leu Gly Phe Ser Gly

195 200 205

Pro His Ser Leu Ala Ala Phe Pro Ala Trp Gly Thr Gln Asp Glu Gly

210 215 220

Thr Leu Glu Phe Thr Leu Thr Thr Gln Ser Arg Gln Ala Pro Leu Ala

225 230 235 240

Phe Gln Ala Gly Gly Arg Arg Gly Asp Phe Ile Tyr Val Asp Ile Phe

245 250 255

Glu Gly His Leu Arg Ala Val Val Glu Lys Gly Gln Gly Thr Val Leu

260 265 270

Leu His Asn Ser Val Pro Val Ala Asp Gly Gln Pro His Glu Val Ser

275 280 285

Val His Ile Asn Ala His Arg Leu Glu Ile Ser Val Asp Gln Tyr Pro

290 295 300

Thr His Thr Ser Asn Arg Gly Val Leu Ser Tyr Leu Glu Pro Arg Gly

305 310 315 320

Ser Leu Leu Leu Gly Gly Leu Asp Ala Glu Ala Ser Arg His Leu Gln

325 330 335

Glu His Arg Leu Gly Leu Thr Pro Glu Ala Thr Asn Ala Ser Leu Leu

340 345 350

Gly Cys Met Glu Asp Leu Ser Val Asn Gly Gln Arg Arg Gly Leu Arg

355 360 365

Glu Ala Leu Leu Thr Arg Asn Met Ala Ala Gly Cys Arg Leu Glu Glu

370 375 380

Glu Glu Tyr Glu Asp Asp Ala Tyr Gly His Tyr Glu Ala Phe Ser Thr

385 390 395 400

Leu Ala Pro Glu Ala Trp Pro Ala Met Glu Leu Pro Glu Pro Cys Val

405 410 415

Pro Glu Pro Gly Leu Pro Pro Val Phe Ala Asn Phe Thr Gln Leu Leu

420 425 430

Thr Ile Ser Pro Leu Val Val Ala Glu Gly Gly Thr Ala Trp Leu Glu

435 440 445

Trp Arg His Val Gln Pro Thr Leu Asp Leu Met Glu Ala Glu Leu Arg

450 455 460

Lys Ser Gln Val Leu Phe Ser Val Thr Arg Gly Ala Arg His Gly Glu

465 470 475 480

Leu Glu Leu Asp Ile Pro Gly Ala Gln Ala Arg Lys Met Phe Thr Leu

485 490 495

Leu Asp Val Val Asn Arg Lys Ala Arg Phe Ile His Asp Gly Ser Glu

500 505 510

Asp Thr Ser Asp Gln Leu Val Leu Glu Val Ser Val Thr Ala Arg Val

515 520 525

Pro Met Pro Ser Cys Leu Arg Arg Gly Gln Thr Tyr Leu Leu Pro Ile

530 535 540

Gln Val Asn Pro Val Asn Asp Pro Pro His Ile Ile Phe Pro His Gly

545 550 555 560

Ser Leu Met Val Ile Leu Glu His Thr Gln Lys Pro Leu Gly Pro Glu

565 570 575

Val Phe Gln Ala Tyr Asp Pro Asp Ser Ala Cys Glu Gly Leu Thr Phe

580 585 590

Gln Val Leu Gly Thr Ser Ser Gly Leu Pro Val Glu Arg Arg Asp Gln

595 600 605

Pro Gly Glu Pro Ala Thr Glu Phe Ser Cys Arg Glu Leu Glu Ala Gly

610 615 620

Ser Leu Val Tyr Val His Arg Gly Gly Pro Ala Gln Asp Leu Thr Phe

625 630 635 640

Arg Val Ser Asp Gly Leu Gln Ala Ser Pro Pro Ala Thr Leu Lys Val

645 650 655

Val Ala Ile Arg Pro Ala Ile Gln Ile His Arg Ser Thr Gly Leu Arg

660 665 670

Leu Ala Gln Gly Ser Ala Met Pro Ile Leu Pro Ala Asn Leu Ser Val

675 680 685

Glu Thr Asn Ala Val Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr

690 695 700

Gly Ala Leu Gln Phe Gly Glu Leu Gln Lys Gln Gly Ala Gly Gly Val

705 710 715 720

Glu Gly Ala Glu Trp Trp Ala Thr Gln Ala Phe His Gln Arg Asp Val

725 730 735

Glu Gln Gly Arg Val Arg Tyr Leu Ser Thr Asp Pro Gln His His Ala

740 745 750

Tyr Asp Thr Val Glu Asn Leu Ala Leu Glu Val Gln Val Gly Gln Glu

755 760 765

Ile Leu Ser Asn Leu Ser Phe Pro Val Thr Ile Gln Arg Ala Thr Val

770 775 780

Trp Met Leu Arg Leu Glu Pro Leu His Thr Gln Asn Thr Gln Gln Glu

785 790 795 800

Thr Leu Thr Thr Ala His Leu Glu Ala Thr Leu Glu Glu Ala Gly Pro

805 810 815

Ser Pro Pro Thr Phe His Tyr Glu Val Val Gln Ala Pro Arg Lys Gly

820 825 830

Asn Leu Gln Leu Gln Gly Thr Arg Leu Ser Asp Gly Gln Gly Phe Thr

835 840 845

Gln Asp Asp Ile Gln Ala Gly Arg Val Thr Tyr Gly Ala Thr Ala Arg

850 855 860

Ala Ser Glu Ala Val Glu Asp Thr Phe Arg Phe Arg Val Thr Ala Pro

865 870 875 880

Pro Tyr Phe Ser Pro Leu Tyr Thr Phe Pro Ile His Ile Gly Gly Asp

885 890 895

Pro Asp Ala Pro Val Leu Thr Asn Val Leu Leu Val Val Pro Glu Gly

900 905 910

Gly Glu Gly Val Leu Ser Ala Asp His Leu Phe Val Lys Ser Leu Asn

915 920 925

Ser Ala Ser Tyr Leu Tyr Glu Val Met Glu Arg Pro Arg His Gly Arg

930 935 940

Leu Ala Trp Arg Gly Thr Gln Asp Lys Thr Thr Met Val Thr Ser Phe

945 950 955 960

Thr Asn Glu Asp Leu Leu Arg Gly Arg Leu Val Tyr Gln His Asp Asp

965 970 975

Ser Glu Thr Thr Glu Asp Asp Ile Pro Phe Val Ala Thr Arg Gln Gly

980 985 990

Glu Ser Ser Gly Asp Met Ala Trp Glu Glu Val Arg Gly Val Phe Arg

995 1000 1005

Val Ala Ile Gln Pro Val Asn Asp His Ala Pro Val Gln Thr Ile

1010 1015 1020

Ser Arg Ile Phe His Val Ala Arg Gly Gly Arg Arg Leu Leu Thr

1025 1030 1035

Thr Asp Asp Val Ala Phe Ser Asp Ala Asp Ser Gly Phe Ala Asp

1040 1045 1050

Ala Gln Leu Val Leu Thr Arg Lys Asp Leu Leu Phe Gly Ser Ile

1055 1060 1065

Val Ala Val Asp Glu Pro Thr Arg Pro Ile Tyr Arg Phe Thr Gln

1070 1075 1080

Glu Asp Leu Arg Lys Arg Arg Val Leu Phe Val His Ser Gly Ala

1085 1090 1095

Asp Arg Gly Trp Ile Gln Leu Gln Val Ser Asp Gly Gln His Gln

1100 1105 1110

Ala Thr Ala Leu Leu Glu Val Gln Ala Ser Glu Pro Tyr Leu Arg

1115 1120 1125

Val Ala Asn Gly Ser Ser Leu Val Val Pro Gln Gly Gly Gln Gly

1130 1135 1140

Thr Ile Asp Thr Ala Val Leu His Leu Asp Thr Asn Leu Asp Ile

1145 1150 1155

Arg Ser Gly Asp Glu Val His Tyr His Val Thr Ala Gly Pro Arg

1160 1165 1170

Trp Gly Gln Leu Val Arg Ala Gly Gln Pro Ala Thr Ala Phe Ser

1175 1180 1185

Gln Gln Asp Leu Leu Asp Gly Ala Val Leu Tyr Ser His Asn Gly

1190 1195 1200

Ser Leu Ser Pro Arg Asp Thr Met Ala Phe Ser Val Glu Ala Gly

1205 1210 1215

Pro Val His Thr Asp Ala Thr Leu Gln Val Thr Ile Ala Leu Glu

1220 1225 1230

Gly Pro Leu Ala Pro Leu Lys Leu Val Arg His Lys Lys Ile Tyr

1235 1240 1245

Val Phe Gln Gly Glu Ala Ala Glu Ile Arg Arg Asp Gln Leu Glu

1250 1255 1260

Ala Ala Gln Glu Ala Val Pro Pro Ala Asp Ile Val Phe Ser Val

1265 1270 1275

Lys Ser Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg Gly

1280 1285 1290

Ala Leu Ala Asp Glu Pro Pro Ser Leu Asp Pro Val Gln Ser Phe

1295 1300 1305

Ser Gln Glu Ala Val Asp Thr Gly Arg Val Leu Tyr Leu His Ser

1310 1315 1320

Arg Pro Glu Ala Trp Ser Asp Ala Phe Ser Leu Asp Val Ala Ser

1325 1330 1335

Gly Leu Gly Ala Pro Leu Glu Gly Val Leu Val Glu Leu Glu Val

1340 1345 1350

Leu Pro Ala Ala Ile Pro Leu Glu Ala Gln Asn Phe Ser Val Pro

1355 1360 1365

Glu Gly Gly Ser Leu Thr Leu Ala Pro Pro Leu Leu Arg Val Ser

1370 1375 1380

Gly Pro Tyr Phe Pro Thr Leu Leu Gly Leu Ser Leu Gln Val Leu

1385 1390 1395

Glu Pro Pro Gln His Gly Ala Leu Gln Lys Glu Asp Gly Pro Gln

1400 1405 1410

Ala Arg Thr Leu Ser Ala Phe Ser Trp Arg Met Val Glu Glu Gln

1415 1420 1425

Leu Ile Arg Tyr Val His Asp Gly Ser Glu Thr Leu Thr Asp Ser

1430 1435 1440

Phe Val Leu Met Ala Asn Ala Ser Glu Met Asp Arg Gln Ser His

1445 1450 1455

Pro Val Ala Phe Thr Val Thr Val Leu Pro Val Asn Asp Gln Pro

1460 1465 1470

Pro Ile Leu Thr Thr Asn Thr Gly Leu Gln Met Trp Glu Gly Ala

1475 1480 1485

Thr Ala Pro Ile Pro Ala Glu Ala Leu Arg Ser Thr Asp Gly Asp

1490 1495 1500

Ser Gly Ser Glu Asp Leu Val Tyr Thr Ile Glu Gln Pro Ser Asn

1505 1510 1515

Gly Arg Val Val Leu Arg Gly Ala Pro Gly Thr Glu Val Arg Ser

1520 1525 1530

Phe Thr Gln Ala Gln Leu Asp Gly Gly Leu Val Leu Phe Ser His

1535 1540 1545

Arg Gly Thr Leu Asp Gly Gly Phe Arg Phe Arg Leu Ser Asp Gly

1550 1555 1560

Glu His Thr Ser Pro Gly His Phe Phe Arg Val Thr Ala Gln Lys

1565 1570 1575

Gln Val Leu Leu Ser Leu Lys Gly Ser Gln Thr Leu Thr Val Cys

1580 1585 1590

Pro Gly Ser Val Gln Pro Leu Ser Ser Gln Thr Leu Arg Ala Ser

1595 1600 1605

Ser Ser Ala Gly Thr Asp Pro Gln Leu Leu Leu Tyr Arg Val Val

1610 1615 1620

Arg Gly Pro Gln Leu Gly Arg Leu Phe His Ala Gln Gln Asp Ser

1625 1630 1635

Thr Gly Glu Ala Leu Val Asn Phe Thr Gln Ala Glu Val Tyr Ala

1640 1645 1650

Gly Asn Ile Leu Tyr Glu His Glu Met Pro Pro Glu Pro Phe Trp

1655 1660 1665

Glu Ala His Asp Thr Leu Glu Leu Gln Leu Ser Ser Pro Pro Ala

1670 1675 1680

Arg Asp Val Ala Ala Thr Leu Ala Val Ala Val Ser Phe Glu Ala

1685 1690 1695

Ala Cys Pro Gln Arg Pro Ser His Leu Trp Lys Asn Lys Gly Leu

1700 1705 1710

Trp Val Pro Glu Gly Gln Arg Ala Arg Ile Thr Val Ala Ala Leu

1715 1720 1725

Asp Ala Ser Asn Leu Leu Ala Ser Val Pro Ser Pro Gln Arg Ser

1730 1735 1740

Glu His Asp Val Leu Phe Gln Val Thr Gln Phe Pro Ser Arg Gly

1745 1750 1755

Gln Leu Leu Val Ser Glu Glu Pro Leu His Ala Gly Gln Pro His

1760 1765 1770

Phe Leu Gln Ser Gln Leu Ala Ala Gly Gln Leu Val Tyr Ala His

1775 1780 1785

Gly Gly Gly Gly Thr Gln Gln Asp Gly Phe His Phe Arg Ala His

1790 1795 1800

Leu Gln Gly Pro Ala Gly Ala Ser Val Ala Gly Pro Gln Thr Ser

1805 1810 1815

Glu Ala Phe Ala Ile Thr Val Arg Asp Val Asn Glu Arg Pro Pro

1820 1825 1830

Gln Pro Gln Ala Ser Val Pro Leu Arg Leu Thr Arg Gly Ser Arg

1835 1840 1845

Ala Pro Ile Ser Arg Ala Gln Leu Ser Val Val Asp Pro Asp Ser

1850 1855 1860

Ala Pro Gly Glu Ile Glu Tyr Glu Val Gln Arg Ala Pro His Asn

1865 1870 1875

Gly Phe Leu Ser Leu Val Gly Gly Gly Leu Gly Pro Val Thr Arg

1880 1885 1890

Phe Thr Gln Ala Asp Val Asp Ser Gly Arg Leu Ala Phe Val Ala

1895 1900 1905

Asn Gly Ser Ser Val Ala Gly Ile Phe Gln Leu Ser Met Ser Asp

1910 1915 1920

Gly Ala Ser Pro Pro Leu Pro Met Ser Leu Ala Val Asp Ile Leu

1925 1930 1935

Pro Ser Ala Ile Glu Val Gln Leu Arg Ala Pro Leu Glu Val Pro

1940 1945 1950

Gln Ala Leu Gly Arg Ser Ser Leu Ser Gln Gln Gln Leu Arg Val

1955 1960 1965

Val Ser Asp Arg Glu Glu Pro Glu Ala Ala Tyr Arg Leu Ile Gln

1970 1975 1980

Gly Pro Gln Tyr Gly His Leu Leu Val Gly Gly Arg Pro Thr Ser

1985 1990 1995

Ala Phe Ser Gln Phe Gln Ile Asp Gln Gly Glu Val Val Phe Ala

2000 2005 2010

Phe Thr Asn Phe Ser Ser Ser His Asp His Phe Arg Val Leu Ala

2015 2020 2025

Leu Ala Arg Gly Val Asn Ala Ser Ala Val Val Asn Val Thr Val

2030 2035 2040

Arg Ala Leu Leu His Val Trp Ala Gly Gly Pro Trp Pro Gln Gly

2045 2050 2055

Ala Thr Leu Arg Leu Asp Pro Thr Val Leu Asp Ala Gly Glu Leu

2060 2065 2070

Ala Asn Arg Thr Gly Ser Val Pro Arg Phe Arg Leu Leu Glu Gly

2075 2080 2085

Pro Arg His Gly Arg Val Val Arg Val Pro Arg Ala Arg Thr Glu

2090 2095 2100

Pro Gly Gly Ser Gln Leu Val Glu Gln Phe Thr Gln Gln Asp Leu

2105 2110 2115

Glu Asp Gly Arg Leu Gly Leu Glu Val Gly Arg Pro Glu Gly Arg

2120 2125 2130

Ala Pro Gly Pro Ala Gly Asp Ser Leu Thr Leu Glu Leu Trp Ala

2135 2140 2145

Gln Gly Val Pro Pro Ala Val Ala Ser Leu Asp Phe Ala Thr Glu

2150 2155 2160

Pro Tyr Asn Ala Ala Arg Pro Tyr Ser Val Ala Leu Leu Ser Val

2165 2170 2175

Pro Glu Ala Ala Arg Thr Glu Ala Gly Lys Pro Glu Ser Ser Thr

2180 2185 2190

Pro Thr Gly Glu Pro Gly Pro Met Ala Ser Ser Pro Glu Pro Ala

2195 2200 2205

Val Ala Lys Gly Gly Phe Leu Ser Phe Leu Glu Ala Asn Met Phe

2210 2215 2220

Ser Val Ile Ile Pro Met Cys Leu Val Leu Leu Leu Leu Ala Leu

2225 2230 2235

Ile Leu Pro Leu Leu Phe Tyr Leu Arg Lys Arg Asn Lys Thr Gly

2240 2245 2250

Lys His Asp Val Gln Val Leu Thr Ala Lys Pro Arg Asn Gly Leu

2255 2260 2265

Ala Gly Asp Thr Glu Thr Phe Arg Lys Val Glu Pro Gly Gln Ala

2270 2275 2280

Ile Pro Leu Thr Ala Val Pro Gly Gln Gly Pro Pro Pro Gly Gly

2285 2290 2295

Gln Pro Asp Pro Glu Leu Leu Gln Phe Cys Arg Thr Pro Asn Pro

2300 2305 2310

Ala Leu Lys Asn Gly Gln Tyr Trp Val

2315 2320

<210> 93

<211> 1210

<212> PRT

<213> Intelligent people

<400> 93

Met Arg Pro Ser Gly Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala

1 5 10 15

Ala Leu Cys Pro Ala Ser Arg Ala Leu Glu Glu Lys Lys Val Cys Gln

20 25 30

Gly Thr Ser Asn Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe

35 40 45

Leu Ser Leu Gln Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn

50 55 60

Leu Glu Ile Thr Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys

65 70 75 80

Thr Ile Gln Glu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val

85 90 95

Glu Arg Ile Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr

100 105 110

Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn

115 120 125

Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu

130 135 140

His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu

145 150 155 160

Ser Ile Gln Trp Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met

165 170 175

Ser Met Asp Phe Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro

180 185 190

Ser Cys Pro Asn Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln

195 200 205

Lys Leu Thr Lys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg

210 215 220

Gly Lys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys

225 230 235 240

Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp

245 250 255

Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro

260 265 270

Thr Thr Tyr Gln Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly

275 280 285

Ala Thr Cys Val Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His

290 295 300

Gly Ser Cys Val Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu

305 310 315 320

Asp Gly Val Arg Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val

325 330 335

Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn

340 345 350

Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp

355 360 365

Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr

370 375 380

Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu

385 390 395 400

Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp

405 410 415

Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln

420 425 430

His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu

435 440 445

Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser

450 455 460

Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu

465 470 475 480

Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu

485 490 495

Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro

500 505 510

Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn

515 520 525

Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly

530 535 540

Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro

545 550 555 560

Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro

565 570 575

Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val

580 585 590

Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp

595 600 605

Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His Pro Asn Cys

610 615 620

Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly

625 630 635 640

Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu

645 650 655

Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met Arg Arg Arg His

660 665 670

Ile Val Arg Lys Arg Thr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu

675 680 685

Val Glu Pro Leu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu

690 695 700

Arg Ile Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser

705 710 715 720

Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu Gly Glu

725 730 735

Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg Glu Ala Thr Ser

740 745 750

Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu Ala Tyr Val Met Ala Ser

755 760 765

Val Asp Asn Pro His Val Cys Arg Leu Leu Gly Ile Cys Leu Thr Ser

770 775 780

Thr Val Gln Leu Ile Thr Gln Leu Met Pro Phe Gly Cys Leu Leu Asp

785 790 795 800

Tyr Val Arg Glu His Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn

805 810 815

Trp Cys Val Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg

820 825 830

Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro

835 840 845

Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu Leu Gly Ala

850 855 860

Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp

865 870 875 880

Met Ala Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp

885 890 895

Val Trp Ser Tyr Gly Val Thr Val Trp Glu Leu Met Thr Phe Gly Ser

900 905 910

Lys Pro Tyr Asp Gly Ile Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu

915 920 925

Lys Gly Glu Arg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr

930 935 940

Met Ile Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys

945 950 955 960

Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp Pro Gln

965 970 975

Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His Leu Pro Ser Pro

980 985 990

Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met Asp Glu Glu Asp Met Asp

995 1000 1005

Asp Val Val Asp Ala Asp Glu Tyr Leu Ile Pro Gln Gln Gly Phe

1010 1015 1020

Phe Ser Ser Pro Ser Thr Ser Arg Thr Pro Leu Leu Ser Ser Leu

1025 1030 1035

Ser Ala Thr Ser Asn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn

1040 1045 1050

Gly Leu Gln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg

1055 1060 1065

Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp

1070 1075 1080

Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln Ser Val Pro

1085 1090 1095

Lys Arg Pro Ala Gly Ser Val Gln Asn Pro Val Tyr His Asn Gln

1100 1105 1110

Pro Leu Asn Pro Ala Pro Ser Arg Asp Pro His Tyr Gln Asp Pro

1115 1120 1125

His Ser Thr Ala Val Gly Asn Pro Glu Tyr Leu Asn Thr Val Gln

1130 1135 1140

Pro Thr Cys Val Asn Ser Thr Phe Asp Ser Pro Ala His Trp Ala

1145 1150 1155

Gln Lys Gly Ser His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln

1160 1165 1170

Gln Asp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys

1175 1180 1185

Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln

1190 1195 1200

Ser Ser Glu Phe Ile Gly Ala

1205 1210

<210> 94

<211> 556

<212> PRT

<213> Intelligent people

<400> 94

Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met

1 5 10 15

Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp

20 25 30

Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln

35 40 45

Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu

50 55 60

Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile

65 70 75 80

Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu

85 90 95

Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr

100 105 110

Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp

115 120 125

Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro

130 135 140

Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala

145 150 155 160

Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro

165 170 175

Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro

180 185 190

Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser

195 200 205

Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser

210 215 220

Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp

225 230 235 240

Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala

245 250 255

Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu

260 265 270

Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly

275 280 285

Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu

290 295 300

Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg

305 310 315 320

Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val

325 330 335

Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu

340 345 350

Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala

355 360 365

Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp

370 375 380

Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly

385 390 395 400

Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu

405 410 415

Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu

420 425 430

Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly

435 440 445

Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu

450 455 460

Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser

465 470 475 480

Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly

485 490 495

Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln

500 505 510

Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala

515 520 525

Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp

530 535 540

Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg

545 550 555

<210> 95

<211> 297

<212> PRT

<213> Intelligent people

<400> 95

Met Thr Thr Pro Arg Asn Ser Val Asn Gly Thr Phe Pro Ala Glu Pro

1 5 10 15

Met Lys Gly Pro Ile Ala Met Gln Ser Gly Pro Lys Pro Leu Phe Arg

20 25 30

Arg Met Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu

35 40 45

Ser Lys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile

50 55 60

Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile

65 70 75 80

Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile

85 90 95

Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys Leu

100 105 110

Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe Ala Ala Ile

115 120 125

Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu Asn Ile Lys Ile Ser

130 135 140

His Phe Leu Lys Met Glu Ser Leu Asn Phe Ile Arg Ala His Thr Pro

145 150 155 160

Tyr Ile Asn Ile Tyr Asn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn

165 170 175

Ser Pro Ser Thr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly

180 185 190

Ile Leu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile

195 200 205

Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys

210 215 220

Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr Ile

225 230 235 240

Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr Ser Ser Gln Pro

245 250 255

Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro Ile Gln Glu Glu Glu Glu

260 265 270

Glu Glu Thr Glu Thr Asn Phe Pro Glu Pro Pro Gln Asp Gln Glu Ser

275 280 285

Ser Pro Ile Glu Asn Asp Ser Ser Pro

290 295

<210> 96

<211> 364

<212> PRT

<213> Intelligent people

<400> 96

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Met Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln

20 25 30

Glu Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro

35 40 45

Tyr Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly

50 55 60

Ala Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln

65 70 75 80

Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro

85 90 95

Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp

100 105 110

Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser

115 120 125

Tyr Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg

130 135 140

Pro Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn

145 150 155 160

Leu Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile

165 170 175

Phe Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr

180 185 190

His Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr

195 200 205

Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu

210 215 220

Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr

225 230 235 240

Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly

245 250 255

Val Val His Gly Ala Ile Gly Gly Ala Gly Val Thr Ala Leu Leu Ala

260 265 270

Leu Cys Leu Cys Leu Ile Phe Phe Ile Val Lys Thr His Arg Arg Lys

275 280 285

Ala Ala Arg Thr Ala Val Gly Arg Asn Asp Thr His Pro Thr Thr Gly

290 295 300

Ser Ala Ser Pro Lys His Gln Lys Lys Ser Lys Leu His Gly Pro Thr

305 310 315 320

Glu Thr Ser Ser Cys Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu

325 330 335

Glu Leu His Tyr Ala Ser Leu Asn Phe His Gly Met Asn Pro Ser Lys

340 345 350

Asp Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln

355 360

<210> 97

<211> 255

<212> PRT

<213> Intelligent people

<400> 97

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

85 90 95

Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu

180 185 190

Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu

195 200 205

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

210 215 220

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

225 230 235 240

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250 255

<210> 98

<211> 256

<212> PRT

<213> mouse

<400> 98

Met Gly Asn Asn Cys Tyr Asn Val Val Val Ile Val Leu Leu Leu Val

1 5 10 15

Gly Cys Glu Lys Val Gly Ala Val Gln Asn Ser Cys Asp Asn Cys Gln

20 25 30

Pro Gly Thr Phe Cys Arg Lys Tyr Asn Pro Val Cys Lys Ser Cys Pro

35 40 45

Pro Ser Thr Phe Ser Ser Ile Gly Gly Gln Pro Asn Cys Asn Ile Cys

50 55 60

Arg Val Cys Ala Gly Tyr Phe Arg Phe Lys Lys Phe Cys Ser Ser Thr

65 70 75 80

His Asn Ala Glu Cys Glu Cys Ile Glu Gly Phe His Cys Leu Gly Pro

85 90 95

Gln Cys Thr Arg Cys Glu Lys Asp Cys Arg Pro Gly Gln Glu Leu Thr

100 105 110

Lys Gln Gly Cys Lys Thr Cys Ser Leu Gly Thr Phe Asn Asp Gln Asn

115 120 125

Gly Thr Gly Val Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Arg

130 135 140

Ser Val Leu Lys Thr Gly Thr Thr Glu Lys Asp Val Val Cys Gly Pro

145 150 155 160

Pro Val Val Ser Phe Ser Pro Ser Thr Thr Ile Ser Val Thr Pro Glu

165 170 175

Gly Gly Pro Gly Gly His Ser Leu Gln Val Leu Thr Leu Phe Leu Ala

180 185 190

Leu Thr Ser Ala Leu Leu Leu Ala Leu Ile Phe Ile Thr Leu Leu Phe

195 200 205

Ser Val Leu Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln

210 215 220

Pro Phe Lys Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser

225 230 235 240

Cys Arg Cys Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu

245 250 255

<210> 99

<211> 254

<212> PRT

<213> cynomolgus monkey

<400> 99

Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu

1 5 10 15

Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Leu Cys Ser Asn Cys Pro

20 25 30

Ala Gly Thr Phe Cys Asp Asn Asn Arg Ser Gln Ile Cys Ser Pro Cys

35 40 45

Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile

50 55 60

Cys Arg Gln Cys Lys Gly Val Phe Lys Thr Arg Lys Glu Cys Ser Ser

65 70 75 80

Thr Ser Asn Ala Glu Cys Asp Cys Ile Ser Gly Tyr His Cys Leu Gly

85 90 95

Ala Glu Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu

100 105 110

Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln

115 120 125

Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys

130 135 140

Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro

145 150 155 160

Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Ala Thr Pro Pro Ala

165 170 175

Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Phe Phe Leu Ala

180 185 190

Leu Thr Ser Thr Val Val Leu Phe Leu Leu Phe Phe Leu Val Leu Arg

195 200 205

Phe Ser Val Val Lys Arg Ser Arg Lys Lys Leu Leu Tyr Ile Phe Lys

210 215 220

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

225 230 235 240

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

245 250

<210> 100

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 100

Gly Gly Gly Gly Ser

1 5

<210> 101

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 101

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 102

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 102

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10

<210> 103

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 103

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

1 5 10

<210> 104

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 104

Gly Ser Pro Gly Ser Ser Ser Ser Gly Ser

1 5 10

<210> 105

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 105

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 106

<211> 20

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 106

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 107

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 107

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210> 108

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 108

Gly Ser Gly Ser Gly Asn Gly Ser

1 5

<210> 109

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 109

Gly Gly Ser Gly Ser Gly Ser Gly

1 5

<210> 110

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 110

Gly Gly Ser Gly Ser Gly

1 5

<210> 111

<211> 4

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 111

Gly Gly Ser Gly

1

<210> 112

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 112

Gly Gly Ser Gly Asn Gly Ser Gly

1 5

<210> 113

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 113

Gly Gly Asn Gly Ser Gly Ser Gly

1 5

<210> 114

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> peptide linker

<400> 114

Gly Gly Asn Gly Ser Gly

1 5

<210> 115

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-H1

<400> 115

Asp Tyr Tyr Met Asn

1 5

<210> 116

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-H2

<400> 116

Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala Ser

1 5 10 15

Val Lys Gly

<210> 117

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-H3

<400> 117

Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr

1 5 10

<210> 118

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-L1

<400> 118

Arg Ala Ser Ser Ser Val Thr Tyr Ile His

1 5 10

<210> 119

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-L2

<400> 119

Ala Thr Ser Asn Leu Ala Ser

1 5

<210> 120

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D)- CDR-L3

<400> 120

Gln His Trp Ser Ser Lys Pro Pro Thr

1 5

<210> 121

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D) VH (3-23A5-1E)

<400> 121

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 122

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (A5H1EL1D) VL (A5-L1D)

<400> 122

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 123

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H24-H29) -CDR-H1

<400> 123

Asp Ser Tyr Met His

1 5

<210> 124

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H24, H25, H27, H28, H29)- CDR-H2

<400> 124

Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe Gln

1 5 10 15

Gly

<210> 125

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H26)- CDR-H2

<400> 125

Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 126

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H24-H29) -CDR-H3

<400> 126

Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr

1 5 10

<210> 127

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L24, L25, L27, L28, L29)- CDR-L1

<400> 127

Arg Ala Ser Ser Ser Val Ser Tyr Met His

1 5 10

<210> 128

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-H26)- CDR-L1

<400> 128

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Met

1 5 10

<210> 129

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L24, L25, L27, L28)- CDR-L2

<400> 129

Ser Thr Ser Asn Leu Ala Ser

1 5

<210> 130

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L26)- CDR-L2

<400> 130

Tyr Thr Ser Asn Leu Ala Ser

1 5

<210> 131

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L29)- CDR-L2

<400> 131

Ser Thr Ser Ser Leu Gln Ser

1 5

<210> 132

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> CEA (MFE-L24, L25, L27, L26, L28, L29)- CDR-L3

<400> 132

Gln Gln Arg Ser Ser Tyr Pro Leu Thr

1 5

<210> 133

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H24

<400> 133

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 134

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H25

<400> 134

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 135

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H26

<400> 135

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 136

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H27

<400> 136

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 137

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H28

<400> 137

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 138

<211> 120

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-H29

<400> 138

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 139

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L24

<400> 139

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 140

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L25

<400> 140

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 141

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L26

<400> 141

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 142

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L27

<400> 142

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 143

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L28

<400> 143

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 144

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> MFE-L29

<400> 144

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 145

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 CDR-H1

<400> 145

Gly Phe Thr Phe Ser Asp Ser Trp Ile His

1 5 10

<210> 146

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 CDR-H2

<400> 146

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

1 5 10 15

Lys Gly

<210> 147

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 CDR-H3

<400> 147

Arg His Trp Pro Gly Gly Phe Asp Tyr

1 5

<210> 148

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 CDR-L1

<400> 148

Arg Ala Ser Gln Asp Val Ser Thr Ala Val Ala

1 5 10

<210> 149

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 CDR-L2

<400> 149

Ser Ala Ser Phe Leu Tyr Ser

1 5

<210> 150

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 CDR-L3

<400> 150

Gln Gln Tyr Leu Tyr His Pro Ala Thr

1 5

<210> 151

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 VH

<400> 151

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 152

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> PD-L1 VL

<400> 152

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 153

<211> 98

<212> PRT

<213> Intelligent people

<400> 153

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Gly Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys

<210> 154

<211> 100

<212> PRT

<213> Intelligent people

<400> 154

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala

20 25 30

Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Thr

100

<210> 155

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-1 VH

<400> 155

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 156

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-2

<400> 156

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Tyr Tyr Gly Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 157

<211> 119

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-3 VH

<400> 157

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Gly Tyr Thr Thr Glu Tyr Ser Ala Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 158

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-4 VH

<400> 158

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 159

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-1A (all back mutations) VH

<400> 159

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 160

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-1C (A93T) VH

<400> 160

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 161

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-23A5-1D (K73) VH

<400> 161

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 162

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-15A5-1 VH

<400> 162

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 163

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-15A5-2 VH

<400> 163

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 164

<211> 121

<212> PRT

<213> Artificial sequence

<220>

<223> 3-15A5-3 VH

<400> 164

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Gly Gly Thr Thr Asp Tyr Ala Ala

50 55 60

Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 165

<211> 95

<212> PRT

<213> Intelligent people

<400> 165

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro

85 90 95

<210> 166

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L1 VL

<400> 166

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 167

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L2 VL

<400> 167

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 168

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L3 VL

<400> 168

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 169

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L4 VL

<400> 169

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 170

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L1A (all back mutations) VL

<400> 170

Gln Thr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 171

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L1B (Q1T2) VL

<400> 171

Gln Thr Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 172

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> A5-L1C (FR2) VL

<400> 172

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 173

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA A5H1EL1D) VHCH1(EE) (20H4.9) -heavy chain HC2 (Fc protrusions)

<400> 173

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Thr Tyr Ile

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Ser Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu

65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Gln His Trp Ser Ser Lys Pro Pro Thr

85 90 95

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 174

<211> 228

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA A5H1EL1D)

<400> 174

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Phe Ile Gly Asn Lys Ala Asn Ala Tyr Thr Thr Glu Tyr Ser Ala

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr

85 90 95

Tyr Cys Thr Arg Asp Arg Gly Leu Arg Phe Tyr Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser

115 120 125

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

130 135 140

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

145 150 155 160

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

165 170 175

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

180 185 190

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

195 200 205

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

210 215 220

Arg Gly Glu Cys

225

<210> 175

<211> 96

<212> PRT

<213> Intelligent people

<400> 175

Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val

1 5 10 15

Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr Tyr Met

20 25 30

His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Trp

35 40 45

Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln Gly

50 55 60

Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr Met Glu

65 70 75 80

Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys Ala Arg

85 90 95

<210> 176

<211> 98

<212> PRT

<213> Intelligent people

<400> 176

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 177

<211> 98

<212> PRT

<213> Intelligent people

<400> 177

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg

<210> 178

<211> 95

<212> PRT

<213> Intelligent people

<400> 178

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro

85 90 95

<210> 179

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA humFE23-L28-H24) VHCH1(EE) (20H4.9) -heavy chain HC2

(Fc protuberance)

<400> 179

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 180

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA humFE23-L28-H24)

<400> 180

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 181

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA humFE23-L28-H28) VHCH1(EE) (20H4.9) -heavy chain HC2

(Fc protuberance)

<400> 181

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 182

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA humFE23-L28-H28)

<400> 182

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 183

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA humFE23-L28-H25) VHCH1(EE) (20H4.9) -heavy chain HC2

(Fc protuberance)

<400> 183

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Leu Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 184

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA humFE23-L28-H25)

<400> 184

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 185

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA humFE 23-L27-H29) VHCH1(EE) (20H4.9) -heavy chain HC2

(Fc protuberance)

<400> 185

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 186

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA humFE 23-L27-H29)

<400> 186

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Thr Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 187

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA humFE23-L27-H28) VHCH1(EE) (20H4.9) -heavy chain HC2

(Fc protuberance)

<400> 187

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 188

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA humFE23-L27-H28)

<400> 188

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 189

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA humFE 23-L27-H26) VHCH1(EE) (20H4.9) -heavy chain HC2

(Fc protuberance)

<400> 189

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 190

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA humFE 23-L27-H26)

<400> 190

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 191

<211> 671

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (CEA humFE23-L27-H24) VHCH1(EE) (20H4.9) -heavy chain HC2

(Fc protuberance)

<400> 191

Glu Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Ser Ser Val Pro Tyr Met

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Pro Glu

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser Ser Ala Ser Thr Lys

100 105 110

Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly

115 120 125

Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

130 135 140

Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

145 150 155 160

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

165 170 175

Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn

180 185 190

Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro

195 200 205

Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val

210 215 220

Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu

225 230 235 240

Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr Trp

245 250 255

Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly Glu

260 265 270

Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser Arg

275 280 285

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu

290 295 300

Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp

305 310 315 320

Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly

325 330 335

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

340 345 350

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

355 360 365

Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

370 375 380

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

385 390 395 400

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

405 410 415

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

420 425 430

Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp Lys

435 440 445

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro

450 455 460

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

465 470 475 480

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

485 490 495

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

500 505 510

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

515 520 525

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

530 535 540

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys

545 550 555 560

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

565 570 575

Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp

580 585 590

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

595 600 605

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

610 615 620

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

625 630 635 640

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

645 650 655

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 192

<211> 227

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (CEA humFE23-L27-H24)

<400> 192

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Asn Glu Gly Thr Pro Thr Gly Pro Tyr Tyr Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val

115 120 125

Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser

130 135 140

Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln

145 150 155 160

Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val

165 170 175

Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu

180 185 190

Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu

195 200 205

Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg

210 215 220

Gly Glu Cys

225

<210> 193

<211> 672

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (PD-L1) VHCH1(EE) (20H4.9) -heavy chain HC2 (Fc protrusions)

<400> 193

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

210 215 220

Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr

225 230 235 240

Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr Tyr

245 250 255

Trp Ser Trp Ile Arg Gln Ser Pro Glu Lys Gly Leu Glu Trp Ile Gly

260 265 270

Glu Ile Asn His Gly Gly Tyr Val Thr Tyr Asn Pro Ser Leu Glu Ser

275 280 285

Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys

290 295 300

Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg

305 310 315 320

Asp Tyr Gly Pro Gly Asn Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg

325 330 335

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

340 345 350

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

355 360 365

Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

370 375 380

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

385 390 395 400

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

405 410 415

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

420 425 430

Pro Ser Asn Thr Lys Val Asp Glu Lys Val Glu Pro Lys Ser Cys Asp

435 440 445

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

450 455 460

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

465 470 475 480

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

485 490 495

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

500 505 510

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

515 520 525

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

530 535 540

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

545 550 555 560

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

565 570 575

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

580 585 590

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

595 600 605

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

610 615 620

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

625 630 635 640

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

645 650 655

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

660 665 670

<210> 194

<211> 225

<212> PRT

<213> Artificial sequence

<220>

<223> VHCL-light chain (PD-L1)

<400> 194

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile

115 120 125

Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val

130 135 140

Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys

145 150 155 160

Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

165 170 175

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

180 185 190

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr

195 200 205

His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

210 215 220

Cys

225

<210> 195

<211> 437

<212> PRT

<213> Artificial sequence

<220>

<223> VLCH1 (PD-L1) -heavy chain HC2 (Fc protrusions)

<400> 195

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser Thr

100 105 110

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

115 120 125

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

130 135 140

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

145 150 155 160

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

165 170 175

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

180 185 190

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

195 200 205

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

210 215 220

Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

225 230 235 240

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

245 250 255

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

260 265 270

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

275 280 285

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

290 295 300

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

305 310 315 320

Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

325 330 335

Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys

340 345 350

Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

355 360 365

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

370 375 380

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

385 390 395 400

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

405 410 415

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

420 425 430

Leu Ser Leu Ser Pro

435

<210> 196

<211> 290

<212> PRT

<213> Intelligent people

<400> 196

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His

225 230 235 240

Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr

245 250 255

Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys

260 265 270

Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu

275 280 285

Glu Thr

290

Claims

1. A bispecific antigen binding molecule comprising:

(a) a first Fab fragment capable of specifically binding to 4-1 BB;

(c) a third Fab fragment capable of specifically binding to 4-1 BB; and

(d) an Fc domain consisting of a first subunit and a second subunit capable of stable association;

wherein the second Fab fragment (b) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the Fab heavy chain of the first Fab fragment (a), which in turn is fused at its C-terminus to the N-terminus of the first Fc domain subunit, and the third Fab fragment (C) is fused at the C-terminus of the Fab heavy chain to the N-terminus of the second Fc domain subunit, and wherein in the second Fab fragment capable of specifically binding to a target cell antigen, (i) variable domains VL and VH are replaced with each other, or (ii) constant domains CL and CH1 are replaced with each other.

2. The bispecific antigen-binding molecule of claim 1, wherein the bispecific antigen-binding molecule provides bivalent binding to 4-1BB and monovalent binding to the target cell antigen.

3. The bispecific antigen binding molecule of claim 1 or 2, wherein the Fc domain consisting of the first and second subunits capable of stable binding is an IgG Fc domain, in particular an IgG1 Fc domain or an IgG4 Fc domain.

4. The bispecific antigen binding molecule of any one of claims 1 to 3, wherein the first subunit of the Fc domain comprises a protuberance and the second subunit of the Fc domain comprises a pore according to a protuberance-into-pore method.

5. The bispecific antigen binding molecule of any one of claims 1 to 4, wherein the Fc domain comprises one or more amino acid substitutions that reduce the binding affinity of the antigen binding molecule to an Fc receptor and/or effector function, in particular the amino acid mutations L234A, L235A and P329G (numbering according to the EU index of Kabat).

6. The bispecific antigen binding molecule of any one of claims 1 to 5, wherein the first and third Fab fragments capable of specific binding to 4-1BB each comprise: heavy chain variable region (V) _H4-1BB) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:1, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:2, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and light chain variable region (V)_L4-1BB) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:4, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:5, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

7. The bispecific antigen binding molecule of any one of claims 1 to 6, wherein the first and third Fab fragments capable of specific binding to 4-1BB each comprise: heavy chain variable region (V)_H4-1BB) comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 7; and light chain variable region (V)_L4-1BB) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 8.

8. The bispecific antigen binding molecule of any one of claims 1 to 7, wherein in the constant domain CL of the first and third Fab fragment capable of specifically binding to 4-1BB, the amino acid at position 124 is substituted with lysine (K) (numbered according to the Kabat EU index) and the amino acid at position 123 is substituted with arginine (R) or lysine (K) (numbered according to the Kabat EU index), and wherein in the constant domain CH1 of the first and third Fab fragment capable of specifically binding to 4-1BB, the amino acid at position 147 is substituted with glutamic acid (E) (numbered according to the Kabat EU index) and the amino acid at position 213 is substituted with glutamic acid (E) (numbered according to the Kabat EU index).

9. The bispecific antigen binding molecule of any one of claims 1 to 8, wherein the second Fab fragment is capable of specifically binding to a target cell antigen selected from the group consisting of: fibroblast Activation Protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), Epidermal Growth Factor Receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, CD33, and PD-L1.

10. The bispecific antigen binding molecule of any one of claims 1 to 9, wherein the second Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to Fibroblast Activation Protein (FAP).

11. The bispecific antigen binding molecule of any one of claims 1 to 10, wherein the Fab fragment capable of specific binding to Fibroblast Activation Protein (FAP) comprises:

(b) Heavy chain variable region (V)_HFAP)Which comprises the following steps: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:15, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:16, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 17; and light chain variable region (V)_LFAP) comprising: (iv) (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:18, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:19, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 20.

12. The bispecific antigen binding molecule of any one of claims 1 to 11, wherein the Fab fragment capable of specific binding to Fibroblast Activation Protein (FAP) comprises:

(b) Heavy chain variable region (V)_HFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 23; and light chain variable region (V) _LFAP) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 24.

13. The bispecific antigen binding molecule of any one of claims 1 to 9, wherein the Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to carcinoembryonic antigen (CEA).

14. The bispecific antigen binding molecule of any one of claims 1 to 9 or claim 13, wherein the Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises:

(a) heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising SEQ I25, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:26, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:28, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:29, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 30; or

(b) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:33, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:34, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35; and light chain variable region (V) _LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:36, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:37, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 38; or

(e) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:115, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:116, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO:117An amino acid sequence; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:118, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:119, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 120; or

(f) Heavy chain variable region (V)_HCEA) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:123, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:124 or SEQ ID NO:125, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 126; and light chain variable region (V)_LCEA) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:127 or SEQ ID NO:128, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:129 or SEQ ID NO:130 or SEQ ID NO:131, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 132.

15. The bispecific antigen binding molecule of any one of claims 1 to 9 or claim 13 or 14, wherein the Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises:

(c) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 47; and light chain variable region (V)_LCEA) comprising SEQ ID NAn amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of O48; or

(d) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; and light chain variable region (V) _LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 56; or

(f) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 55; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 56; or

(g) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 133; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 143; or

(h) Heavy chain variable region (V)_HCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 137; and light chain variable region (V)_LCEA) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID No. 143.

16. According toThe bispecific antigen binding molecule of any one of claims 1 to 9 or claim 13 or 14, wherein the Fab fragment capable of specific binding to carcinoembryonic antigen (CEA) comprises: heavy chain variable region (V)_HCEA) comprising the amino acid sequence of SEQ ID NO 133, SEQ ID NO 134, SEQ ID NO 135, SEQ ID NO 136, SEQ ID NO 137 or SEQ ID NO 138; and light chain variable region (V)_LCEA) comprising the amino acid sequence of SEQ ID NO 139, 140, 141, 142, 143 or 144.

17. The bispecific antigen binding molecule of any one of claims 1 to 9, wherein the Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to CD 19.

18. The bispecific antigen binding molecule of any one of claims 1 to 9 or claim 17, wherein the Fab fragment capable of specific binding to CD19 comprises:

19. The bispecific antigen binding molecule of any one of claims 1 to 9 or claim 17 or 18, wherein the Fab fragment capable of specifically binding to CD19 comprises: heavy chain variable region (V)_HCD19) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID No. 63; and light chain variable region (V)_LCD19) comprising ammonia that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO. 64An amino acid sequence.

20. The bispecific antigen binding molecule of any one of claims 1 to 9, wherein the Fab fragment capable of specifically binding to a target cell antigen is a Fab fragment capable of specifically binding to PD-L1.

21. The bispecific antigen binding molecule of any one of claims 1 to 9 or claim 20, wherein the Fab fragment capable of specifically binding to PD-L1 comprises:

(a) heavy chain variable region (V)_HPD-L1) comprising: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO:145, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO:146, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 147; and light chain variable region (V)_LPD-L1) comprising: (iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO:148, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:149, and (vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 150.

22. The bispecific antigen binding molecule of any one of claims 1 to 9 or claim 20 or 21, wherein the Fab fragment capable of specifically binding to PD-L1 comprises: heavy chain variable region (V)_HPD-L1) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 152; and light chain variable region (V)_LPD-L1) comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 153.

23. A polynucleotide encoding the bispecific antigen binding molecule of any one of claims 1 to 22.

24. A vector comprising the polynucleotide of claim 23.

25. A host cell comprising the polynucleotide of claim 23 or the vector of claim 24.

26. A method of producing the bispecific antigen binding molecule of any one of claims 1 to 22, the method comprising culturing the host cell of claim 25 under conditions suitable for expression of the bispecific antigen binding molecule.

27. A pharmaceutical composition comprising the bispecific antigen binding molecule of any one of claims 1 to 22 and at least one pharmaceutically acceptable excipient.

28. The bispecific antigen binding molecule of any one of claims 1 to 22 or the pharmaceutical composition of claim 27 for use as a medicament.

29. The bispecific antigen binding molecule of any one of claims 1 to 22 or the pharmaceutical composition of claim 27, for use in the treatment of cancer.

30. The bispecific antigen binding molecule of any one of claims 1 to 22, for use in the treatment of cancer, wherein the bispecific antigen binding molecule is administered in combination with a chemotherapeutic agent, radiation therapy and/or other agent for cancer immunotherapy.

31. Use of the bispecific antigen binding molecule of any one of claims 1 to 22 or the pharmaceutical composition of claim 27 in the manufacture of a medicament for the treatment of cancer or an infectious disease.

32. A method of inhibiting growth of a tumor cell in an individual, the method comprising administering to the individual an effective amount of the bispecific antigen binding molecule of any one of claims 1 to 22 or the pharmaceutical composition of claim 27 to inhibit growth of the tumor cell.

33. A method of treating cancer or an infectious disease, the method comprising administering to the individual a therapeutically effective amount of the bispecific antigen binding molecule of any one of claims 1 to 22 or the pharmaceutical composition of claim 27.